Novel probiotic whey cheese with immobilized lactobacilli on casein

Accepted Manuscript Novel probiotic whey cheese with immobilized lactobacilli on casein Dimitra Dimitrellou, Panagiotis Kandylis, Yiannis Kourkoutas, Maria Kanellaki PII:

S0023-6438(17)30598-4

DOI:

10.1016/j.lwt.2017.08.028

Reference:

YFSTL 6450

To appear in:

LWT - Food Science and Technology

Received Date: 27 November 2016 Revised Date:

14 July 2017

Accepted Date: 9 August 2017

Please cite this article as: Dimitrellou, D., Kandylis, P., Kourkoutas, Y., Kanellaki, M., Novel probiotic whey cheese with immobilized lactobacilli on casein, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.08.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Novel probiotic whey cheese with immobilized lactobacilli on

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casein

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Short title: Probiotic whey cheese with lactobacilli

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Dimitra Dimitrellou1,2*, Panagiotis Kandylis1,2, Yiannis Kourkoutas1, Maria

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Kanellaki2

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Applied Microbiology and Molecular Biology Research Group, Department of Molecular

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Biology and Genetics, Democritus University of Thrace, Alexandroupolis, 68100, Greece 2

Food Biotechnology Research Group, Department of Chemistry, University of Patras, Patra

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26500, Greece

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E-mail.:[email protected]

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Corresponding author. Dr. Dimitra Dimitrellou; Tel.: +306946489978.

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ACCEPTED MANUSCRIPT ABSTRACT

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In the present study, the use of thermally-dried immobilized lactobacilli (Lactobacillus casei

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ATCC 393 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842) on casein was evaluated

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for the production of a novel dried whey cheese. Cheeses with thermally-dried free

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lactobacilli cells and with no starter culture were also produced, for comparison reasons.

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The main physicochemical and microbiological characteristics of a novel whey cheese were

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determined during ripening at 20oC until day 60. The results clearly demonstrated that the

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use of free and immobilized on casein L. casei and L. bulgaricus positively affected the

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production of dried whey cheeses. More specifically, their use led to novel whey cheeses

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with improved quality, as resulted by sensory evaluation and SPME GC/MS analysis of

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aroma, extended preservation time and protection from spoilage and pathogens. Whey

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cheeses microbiota was dominated by lactobacilli and mesophilic and thermophilic

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lactococci. Counts of coliforms, enterobacteria and staphylococci were significantly reduced

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in whey cheese produced with thermally-dried lactobacilli cultures. The use of cultures, like

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those proposed in the present study, may have a great potential in dairy technology for the

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conversion of low quality whey cheeses to novel probiotic foods with improved

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characteristics.

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Keywords: L. casei, L. bulgaricus, thermal drying, GC/MS

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ACCEPTED MANUSCRIPT 1. Introduction

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Cheese whey is the liquid waste of the dairy industry remaining after milk coagulation and

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removal of the curd during cheese production. Cheese production is continuously increasing

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with a rate of 2% per year and therefore large quantities of whey, approximately 120 million

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tons per year, are also produced representing a serious environmental problem

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(Kaminarides, 2015). Whey utilization is constantly increasing and currently about 50% of

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total whey produced is transformed into value-added products such as whey powder, whey

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protein, biofuels, biopolymers, electricity, single cell protein, probiotic dairy products,

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prebiotics, ethanol etc (Dimitrellou, Kourkoutas, Koutinas, & Kanellaki, 2009; Dimitrellou,

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Kourkoutas, Banat, Marchant, & Koutinas, 2007; Koutinas et al., 2009; Prazeres, Carvalho, &

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Rivas, 2012; Sen, Bhattacharjee, & Bhattacharya, 2016; Yadav et al., 2014). However, large

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volumes of whey still remain untreated and are discarded in the environment. The chemical

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composition of whey differs according to the types of cheese produced and especially the

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kind of milk used for cheese production. In fact, cheese whey contains water 93-94%, lactose

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4.5-6.0%, proteins 0.6-1.1%, minerals 0.8-1.0%, lactic acid 0.05-0.9% and fats 0.06-0.5%

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(Prazeres et al., 2012).

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Traditionally, in the Mediterranean region a large portion of whey is used for the production

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of whey cheeses (Pintado & Malcata, 2000). The production of whey cheeses is based on

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thermal (above 85oC) denaturation and coagulation of the water soluble milk proteins

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present in whey (a-lactalbumin and b-lactoglobulin). Of all Greek whey cheeses, Myzithra, a

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soft cheese produced from the whey of Feta cheese or hard cheeses, such as Kefalotyri and

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Graviera, is produced in the largest quantity (Kaminarides, 2015). In the market, two types of

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Myzithra are available: fresh Myzithra, with fat content in dry matter of about 50% and a

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moisture content that is not greater than 70%, but with limited shelf life and dry Myzithra,

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with the same fat content in dry matter and a moisture content that is not greater than 40%

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ACCEPTED MANUSCRIPT (Anifantakis, 1991). Due to the nature of their production at high temperatures, no culture is

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added in whey cheeses and therefore they are susceptible to spoilage.

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Lactobacillus casei ATCC 393 is a strain with satisfying technological characteristics and

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therefore has numerous applications in the production of food products, such as cheese

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(Dimitrellou, Kandylis, Sidira, Koutinas, & Kourkoutas, 2014a), sausages (Sidira, Dimitrellou,

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Kanellaki, & Kourkoutas, 2010a), bread (Plessas et al., 2007), fermented milks (Dimitrellou et

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al., 2016a) and yogurts (Dimitrellou, Kandylis, & Kourkoutas, 2014b). In addition, recent

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studies have demonstrated that L. casei ATCC 393 has beneficial probiotic effects, such as

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removal of cholesterol (Lye, Rusul, & Liong, 2010), reduction of osteoporosis risk (Kim et al.,

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2009), tumor-inhibitory, anti-proliferative and pro-apoptotic effects (Tiptiri-Kourpeti et al.,

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2016). On the other hand, Lactobacillus delbrueckii ssp. bulgaricus is widely used in yogurt

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production in combination with Streptococcus thermophilus. More specifically, L. bulgaricus

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ATCC 11842 is a strain that has been used in the production of several foods like bread

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(Plessas et al., 2008) and cheese (Katechaki, Solomonidis, Bekatorou, & Koutinas, 2010).

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Moreover, this strain has presented anti-bacterial and anti-adherence effects against

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Escherichia coli in vitro (Abedi, Akbari, & Jafarian-Dehkordi, 2013).

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It is well established that cell immobilization enhances the viability of cultures during food

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production, processing, storage and simulated gastrointestinal conditions (Dimitrellou et al.,

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2016a; Dimitrellou, Kandylis, & Kourkoutas, 2016b). Milk proteins are characterized as

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natural vehicles for the delivery of bioactives, including probiotics, due to their structural

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and physicochemical properties (Livney, 2010). Milk proteins have been used for

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microencapsulation and delivery of bioactive compounds and probiotics (Anal & Singh, 2007;

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Chen & Subirade, 2006; Hébrard et al., 2006; Heidebach, Först, & Kulozik, 2009).

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Furthermore, casein and whey proteins have been used successfully as immobilization

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support of probiotic cells for the production of several dairy products (Dimitrellou et al.,

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ACCEPTED MANUSCRIPT 2016b; Dimitrellou, Kandylis, Kourkoutas, Koutinas, & Kanellaki, 2015; Dimitrellou et al.,

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2014b; Dimitrellou et al., 2009; Sidira et al., 2017).

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The aim of the present study was to evaluate the feasibility of producing a novel whey

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cheese, with extended shelf life, using probiotic lactobacilli immobilized on casein in its

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production. The immobilized cells were thermally dried aiming to produce a more stable and

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ready to use product, complying with the industrial needs, but with low cost. The effect of

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lactobacilli on physicochemical and microbiological characteristics of novel whey cheeses

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was evaluated along with the profile of major volatile compounds which are responsible for

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the aroma of new products.

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2. Materials and methods

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2.1 Strains

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Probiotic cultures of Lactobacillus casei ATCC 393 and Lactobacillus delbrueckii ssp.

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bulgaricus ATCC 11842 (DSMZ, Braunschweig, Germany) were grown at 37°C for 72 h on de

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Man, Rogosa, and Sharpe (MRS) broth (Fluka, Buchs, Switzerland).

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2.2 Cell immobilization on casein

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For cell immobilization (Dimitrellou et al., 2015), 3 g (wet weight) of either Lactobacillus

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casei ATCC 393 or Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 were added to 1.5 L

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of commercial pasteurized bovine milk (0% fat). The mixture was heated to 37oC,

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commercial rennet (0.01%) was added, and left undisturbed for 1 h for casein precipitation.

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Subsequently, the curd was cut in cubes (acme ≈1 cm), left undisturbed for 10 min and was

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then cloth filtered at room temperature (18–22oC). Casein-supported biocatalyst was

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thermally-dried prior use.

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ACCEPTED MANUSCRIPT 2.3 Thermal-drying procedure

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Casein-supported biocatalysts and free cultures were dried in a chamber equipped with air

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circulation (J.P. Selecta, Spain) at 38oC until constant weight (≈24 h) (Dimitrellou et al.,

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2008).

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2.4 Novel dried whey cheese production

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Fresh whey cheese was provided by a local dairy industry (AVIGAL S.A. Valmantoura, Achaia,

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Greece) and produced using sweet whey derived after Feta-cheese production. Ewe’s milk

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(2.3%) and salt (0.3%) were added in the whey and the mixture was heated to 95oC for 10-15

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min until coagulation and the coagulant was then cloth filtered. The thermally-dried cultures

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were suspended in whey for 2 h at 30oC prior to incorporation into the cheese curd.

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Specifically, for the production of dried whey cheese using thermally-dried immobilized cells

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on casein (ILC and ILB), 75 g of thermally-dried immobilized cells were homogenized with

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100 mL whey and mixed with the cheese curd (1500 g) during cloth filtration, while for

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production of dried whey cheese using thermally-dried free cells (FLC and FLB), 45 ml of

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whey containing 4.5 g of thermally dried free cells were sprayed at cheese curd (1500 g)

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during cloth filtration (Dimitrellou et al., 2009). The produced cheese samples were ripened

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at 20oC (relative humidity 61%) and ripening was monitored for up to 60 days. Traditional

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dried whey cheese without culture (C) was also produced in order to allow comparison of

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results. All treatments were carried out in triplicate and the mean values are presented.

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Duplicate samples from each treatment were collected at various intervals (1, 4, 15, 30, 45

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and 60 days) and were subjected to physicochemical and microbiological analysis.

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2.5 Physicochemical analysis

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Cheese samples (20 g each) were macerated with warm water (40°C) to produce a total

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volume of 210 mL. Each sample was then filtered and the filtrate was used for lactic acid,

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ACCEPTED MANUSCRIPT ethanol, and residual sugar determination (AOAC International, 1995). Moisture content, ash

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content, and acidity (expressed as lactic acid) of cheese samples were determined according

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to AOAC International (1995). Residual sugar (lactose, glucose, and galactose) were

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determined using HPLC, as described previously (Dimitrellou et al., 2009). All analyses were

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carried out in triplicate and the mean data are presented.

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2.6 Solid-Phase Microextraction GC-MS Analysis

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Cheese samples ripened for 60 days were used to study the volatile composition by solid-

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phase microextraction (SPME) GC-MS analysis. The procedure described in a previous study

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(Dimitrellou et al., 2009) was followed. More specifically semi-quantification of volatile

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compounds was carried out using methyl octanoate (Sigma–Aldrich, Poole, UK) diluted in

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pure hexane as an internal standard (IS) at various concentrations (1.25, 12.5, 125 and 1250

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mg/kg of cheese). The volatile compounds were semi-quantified by dividing the peak areas

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of the compounds of interest by the peak area of the IS and multiplying this ratio by the

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initial concentration of the IS (expressed as mg/kg). The peak areas were measured from the

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full scan chromatograph using total ion current (TIC). Identification was carried out by

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comparing the retention times and mass spectra of volatiles to those of authentic

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compounds generated in the laboratory, by mass spectra obtained from NIST107, NIST21,

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and SZTERP libraries [CLASS 5000 software of GC-17A/QP-5050A (GC-MS); Shimadzu Corp.,

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Kyoto, Japan], and by determining Kovats’ retention indices and comparing them with those

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reported in the literature (Bianchi, Careri, Mangia, & Musci, 2007; Dimitrellou et al., 2015;

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Dimitrellou et al., 2009; Kandylis, Drouza, Bekatorou, & Koutinas, 2010). Each determination

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was carried out in triplicate and the mean data are presented.

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2.7. Microbiological analyses

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Microbiological analysis was carried out as described previously (Dimitrellou et al., 2009).

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ACCEPTED MANUSCRIPT 2.8. Sensory evaluation of whey cheeses

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The novel dried whey cheeses produced with thermally dried free and immobilized cultures

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and without culture were tested for their sensory characteristics, as described in a previous

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study (Dimitrellou et al., 2009).

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2.9. Statistical analysis

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All experiments were carried out in triplicate. Significance was established at P < 0.05. The

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results were analyzed for statistical significance with ANOVA, and Tukey honest significant

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difference (HSD) test was used to determine significant differences between the results;

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coefficients, ANOVA tables, and significance (P < 0.05) were computed using Statistica

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version 5.0 (StatSoft Inc., Tulsa, OK).

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3. Results and discussion

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3.1. Production of novel probiotic whey cheeses with immobilized L. casei and L.

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bulgaricus on casein

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Whey cheeses were produced using the traditional method of a local dairy industry. Since

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traditionally whey cheeses are produced without any starter culture, the selection of

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potential probiotic cultures is a very crucial step. Therefore, in the present study, two strains

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with numerous applications in dairy products, namely L. casei ATCC 393 and L. bulgaricus

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ATCC 11842, were used as potential probiotic strains. Lactobacillus casei ATCC 393 was

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selected based also on its capability to survive the harsh conditions of the GI tract as was

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proved both by in vitro, using simulated GI conditions (Dimitrellou et al., 2016a), and in vivo

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studies, using Wistar rats (Sidira et al., 2010b). Casein was selected as immobilization

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support, since milk proteins act as buffering agents in vivo, protecting bacterial strains at GI

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ACCEPTED MANUSCRIPT tract (Charteris, Kelly, Morelli, & Collins, 1998). Furthermore, both strains were used in dried

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form in order to be in accordance to the industrial needs for ready to use cultures that will

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be also capable to be stored for a short period. Indeed, previous studies have proved that

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both strains are capable to survive the harsh conditions during drying either in free or

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immobilized form (Dimitrellou et al., 2016a; Dimitrellou et al., 2016b; Koutinas et al., 2009).

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Whey cheeses without culture using the traditional method were also produced for

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comparison reasons.

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3.2. Physicochemical characteristics of novel probiotic whey cheeses

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The results concerning the effect of the thermally dried immobilized and free lactobacilli on

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the physicochemical characteristics of dried whey cheese during ripening are summarized in

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Table 1. The thermally dried cultures, cell immobilization and ripening time affected

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significantly (P<0.05) all parameters analyzed apart from glucose/galactose, moisture and

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ash content, which were affected significantly (P<0.05) only by the ripening time.

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In general, lactose content was significantly (P<0.05) lower in whey cheese sample produced

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with either free or immobilized lactobacilli compared to traditional whey cheese. This trend

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was expected due to the presence of culture during ripening. No significant differences were

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observed in the case of sugars between whey cheeses with L. casei and L. bulgaricus either

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in free or immobilized form and these values were similar to those in previous studies with

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dried whey cheese using kefir culture (Dimitrellou et al., 2009). In the case of pH, traditional

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dried whey cheese produced without any culture presented values similar to other dried

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whey cheeses like “Urda” (Pappa, Samelis, Kondyli, & Pappas, 2016), but in all cases, these

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values were significant higher than those of whey cheeses produced with lactobacilli. As a

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consequence, the acidity of cheeses without culture was significant lower than in cheeses

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with lactobacilli. In addition, the use of immobilized cells resulted in significant higher values

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of acidity compare to cheeses with free cells of lactobacilli. The same trend was observed

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ACCEPTED MANUSCRIPT when dried whey cheeses were produced with free and immobilized kefir culture

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(Dimitrellou et al., 2009). This is probably due to the improved adaption of immobilized cells

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and higher fermentative activity. Moisture content was not affected by the use of cultures,

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but was significantly (P<0.05) reduced during ripening and therefore the ash content was

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significantly (P<0.05) increased, especially from day 1 to 4 due to salting of the cheeses. The

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moisture content of cheeses of the present study was similar to a previous study with kefir

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culture (Dimitrellou et al., 2009), but significant higher compared to “Urda”, another dried

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whey cheese (Pappa et al., 2016). The different technological parameters may explain the

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observed differences.

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3.3. Microbiological analysis of novel probiotic whey cheeses

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The association of the microbial groups examined during ripening of the novel dried whey

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cheeses is presented in Tables 2 and 3. Due to the production process of whey cheeses that

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requires high temperatures above 85oC, these cheeses are practically free of bacteria after

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production (Pintado & Malcata,2000). However, handling of cheese curd after production

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and the equipment are responsible for cross-contamination with many microorganisms

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(Kalogridou-Vassiliadou, Tzanetakis, & Litopoulou-Tzanetaki, 1994; Lioliou, Litopoulou-

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Tzanetaki, Tzanetakis, & Robinson, 2001; Pintado & Malcata, 2000). In the present study, the

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numbers of lactobacilli were significant higher in the case of whey cheeses with free or

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immobilized L. casei and L. bulgaricus, as expected, Of note, their numbers also increased

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significantly in the case of traditional whey cheeses without cultures, as observed in other

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whey cheeses (Papaioannou, Chouliara, Karatapanis, Kontominas, & Savvaidis, 2007; Pappa

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et al., 2016). No significant differences were observed in the numbers of mesophilic and

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thermophilic lactococci among all whey cheeses of the present study. Their presence in

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cheeses is due to non starter lactic acid bacteria and cross-contamination after cheese

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production. A reduction in their numbers at the end of ripening may be attributed to the

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ACCEPTED MANUSCRIPT high salt and low moisture content (Psoni, Tzanetakis, & Litopoulou-Tzanetaki, 2006). The

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high numbers of yeast at the first day of ripening in all cheeses (≈ 5 log cfu/g) is probably due

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to contaminations from cheese making equipment and environment (Viljoen, 2001).

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However, their numbers were reduced during ripening, in accordance to results previously

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published concerning “Urda” cheese (Pappa et al., 2016). This reduction was more rapid in

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the case of whey cheeses with free or immobilized L. casei and L. bulgaricus. The numbers of

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coliforms, enterobacteria and staphylococci were increased during ripening and after the

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15th day a reduction was observed. In cheeses with lactobacilli their numbers at the end of

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ripening were significant (P<0.05) lower compared to traditional whey cheese with no

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culture. This may be attributed to the lower pH values, lower moisture content and higher

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acidity that do not favor their growth. The significantly (P<0.05) reduced numbers of

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coliforms, staphylococci and enterobacteria in dried whey cheeses produced with free or

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immobilized L. casei and L. bulgaricus compared to cheeses without culture and to other

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whey (Dimitrellou et al., 2009; Kalogridou-Vassiliadou et al., 1994), other Greek (Gerasi,

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Litopoulou-Tzanetaki, & Tzanetakis, 2003; Hatzikamari, Litopoulou-Tzanetaki, & Tzanetakis,

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1999; Nikolaou, Litopoulou-Tzanetaki, Tzanetakis, & Robinson, 2002) and Portuguese (Pinho

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et al., 2004) cheeses, suggested the development of microbial association, leading to

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repression of spoilage and pathogenic bacteria. Although pathogenesis or spoilage has not

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been reported for all staphylococci (Kourkoutas et al., 2006) and coliforms should not be

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associated with pathogens (Trmčić et al., 2016), the reduction in their numbers is a very

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important observation for the production process and sensorial characteristics of the novel

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whey cheeses.

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In order to evaluate the effect of lactobacilli on preservation of novel dried whey cheeses, all

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cheeses left at 20oC without packaging until spoilage was observed on their surface. More

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specifically, sparse surface greenish spots were observed in traditional dried whey cheeses

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after 27 days of ripening, while in novel whey cheeses produced with free L. casei and L.

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ACCEPTED MANUSCRIPT bulgaricus after 42 and 43 days, respectively. The use of immobilized cells further increased

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the preservation time of cheeses with spoilage observed after 51-52 days. Similar significant

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(P<0.05) increase of the preservation time of whey cheeses as was also observed in the case

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of dried whey cheeses with kefir culture (Dimitrellou et al., 2009). The spots were probably

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molds, which are often observed in the surface of dried whey cheeses during ripening, but

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they are commonly harmless. It is a usual practice to remove them, by cutting at least ¼ inch

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beneath the mold before consumption or packaging (Zerfiridis, 2001).

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3.4. Aroma related compounds of novel probiotic whey cheeses

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Whey cheese samples produced with L. casei and L. bulgaricus, either in free (FLC and FLB)

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or immobilized form (ILC and ILB), were analyzed using SPME GC/MS technique and

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compared to whey cheeses produced with no starter culture (C) in order to evaluate the

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effect of lactobacilli on the production of major volatile compounds. In total, 49 compounds

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were detected, from which 25 in C sample, 33 in FLC, 25 in FLB, 33 in ILC and 29 in ILB. These

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compounds included esters, organic acids, alcohols, carbonyl compounds and lactones.

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Organic acids were the most abundant volatile group isolated in the headspace of whey

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cheeses (Table 4), which is in accordance with the results of a previous study concerning

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dried whey cheese (Dimitrellou et al., 2009). Indeed, fatty acids have been proved important

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components of the flavor of many cheese types (Curioni & Bosset, 2002). In general, the

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content of organic acids was higher in whey cheeses with L. casei and L. bulgaricus and

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especially in those produced with immobilized cells (ILC and ILB). Τhe acids with the higher

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concentrations were hexanoic, octanoic and decanoic acid, which were considered as major

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flavor components of other cheese types, such as aged Cheddar, Grana Padano and Roncal

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cheese (Curioni & Bosset, 2002). Dodecanoic acid was also detected in high concentrations

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in all cheeses of the present study. However, it plays a minor role in cheese flavor, due to its

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low perception threshold. Branched-chain fatty acids, like 2-methyl and 3-methyl butanoic

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ACCEPTED MANUSCRIPT acids, detected only in FLC and ILB, are considered important compounds of goat and sheep

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cheeses, and together contribute to the very-ripened-cheese aroma (Yvon & Rijnen, 2001).

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Alcohols were the second more abundant volatile group of whey cheeses. The alcohol with

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the higher concentration detected in all whey cheese samples was phenylethanol. This

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compound is among the most odorous alcohols with a pleasant rose flower notes (Curioni &

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Bosset, 2002) originating from phenylalanine through microbial catabolism (Ziino, Condurso,

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Romeo, Giuffrida, & Verzera, 2005). 3-Methyl-1-butanol, detected in all whey cheeses apart

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from ILC and ILB, provides a pleasant aroma of fresh cheese and originated from leucine.

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In total, 12 esters were detected .Seven of them belong to the group of ethyl esters, which

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are considered important compounds for the flavor of cheeses, being responsible for the

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fruity character (Curioni & Bosset, 2002). 2-Phenylethyl acetate, detected in all cheeses, is

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one of the most important flavor ester in cheeses, providing floral and rose-like attributes

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(Molimard & Spinnler, 1996), especially in Camembert cheese (Kubíčková & Grosch, 1997).

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The carbonyl compounds detected belong to aldehydes and ketones group. Their

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concentrations were similar in all whey cheeses, but cheeses with free cells of lactobacilli

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(FLC and FLB) presented slight higher concentrations. Aldehydes are not present in high

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concentrations, because they are rapidly reduced to alcohols or oxidized to acids (Curioni &

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Bosset, 2002). Only two aldehydes were detected in the whey cheeses of the present study,

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namely 3-methyl butanal and benzaldehyde. Benzaldehyde detected in all cheeses is

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described as having an odor of bitter almond (Molimard & Splinnler, 1996). 3-Methyl

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butanal detected only in ILC, probably originates from leucine degradation and has been

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detected in other cheeses, such as Camambert, aged Cheddar and Feta-type cheese (Curioni

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& Bosset, 2002; Dimitrellou et al., 2014a). Ketones are common flavor compounds in most

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dairy products and especially methyl ketones like 2-octanone and 2-undecanone, which are

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known for their fruity, floral and musty notes, and 2-heptanone for its blue cheese notes

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ACCEPTED MANUSCRIPT (Curioni & Bosset, 2002). In the present study these methyl ketones were detected in all

306

cheese samples. 3-Hydroxy-2-butanone (acetoin) was present only in cheeses with

307

lactobacilli and this is attributed to the fact that it originates from citrate metabolism by the

308

action of lactic acid bacteria (McSweeney and Fox, 2004).

309

Lactones are cyclic esters and among them γ- and δ-lactones are considered important

310

compounds for the final aroma of several cheeses, such as Camembert (Curioni & Bosset,

311

2002). They are known to have fruity aromas like peach, apricot and coconut and especially

312

δ-lactones have low threshold limits (Vagenas & Roussis, 2012). In the present study, γ- and

313

δ- decalactone and dodecalactone were detected in almost all whey cheeses. Higher

314

concentrations of lactones were detected in whey cheeses with L. casei (FLC and ILC). This

315

trend was also reported in Feta-type cheeses produced with free or immobilized on whey

316

proteins L. casei cells (Dimitrellou et al., 2014a).

317

The total concentration of each aroma group per whey cheese is summarized in Table 5. In

318

the case of esters, FLC and ILB presented significantly higher (P<0.05) concentrations

319

compared to other whey cheeses. Organic acids was the group with the highest

320

concentrations in all whey cheeses. No significant differences were observed between the

321

control (C) and whey cheeses with L. casei (FLC and ILC), while in the case of whey cheeses

322

with L. bulgaricus (FLB and ILB) their content was significantly (P<0.05) higher. Whey cheeses

323

with L. casei presented significantly higher (P<0.05) concentrations of lactones compared to

324

other whey cheeses. Finally, total concentrations of aroma compounds were higher in

325

cheeses with L. bulgaricus (ILB > FLB) followed by those with L. casei (FLC > ILC) and control

326

whey cheese (C).

327

3.5. Preliminary sensory evaluation of novel whey cheeses

AC C

EP

TE D

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305

14

ACCEPTED MANUSCRIPT The whey cheeses produced using thermally dried immobilized and free L. casei or L.

329

bulgaricus were compared with traditional whey cheeses without culture in regard to their

330

sensory characteristics (data not shown). Generally, the use of lactobacilli cultures positively

331

affected the flavor of dried whey cheeses leading to significantly (P<0.05) higher values

332

compared to traditional cheeses. No significant differences were observed between cheeses

333

with free or immobilized cells or between different lactobacilli used.

334

4. Conclusions

335

The results of the present study clearly demonstrated that the use of free and immobilized L.

336

casei and L. bulgaricus on casein positively affected the production of dried whey cheeses.

337

More specifically, their use led to novel whey cheeses with improved quality, as resulted by

338

sensory evaluation and SPME GC/MS analysis of aroma, extended preservation time and

339

protection from spoilage and pathogens. The use of such cultures may have a great potential

340

in dairy technology for the conversion of low quality whey cheeses to novel probiotic foods

341

with improved characteristics. The proposed technology may be adapted to the production

342

of other whey cheeses, as well, while the low cost of thermal drying process is in accordance

343

with the industrial needs for ready to use dried cultures.

EP

TE D

M AN U

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Table 1. Effect of different lactobacilli cultures on physicochemical characteristic of novel dried whey cheeses during ripening.

C

1

FLC

3.98±0.16

4

2.94±0.10

15

b,C

1.08±0.07

30

0.41±0.05

45

Tr

60

Tr

a,B

b,A

0.26±0.04

(g/100g cheese)

4

0.11±0.02

0.19±0.02

a,A

A

0.39±0.04

0.19±0.04

B A

Tr

Tr

45

Tr

Tr

60

Tr

Tr

6.60±0.10

b,B

4

6.27±0.08

b,B

15

5.60±0.04

c,A

30

5.54±0.06

b,A

45

5.35±0.02

d,A

60

5.25±0.06

acid/100g

15

cheese)

30 45

6.30±0.07

5.10±0.07

0.26±0.06

a,B

0.50±0.07

AB

0.54±0.05

a,B

0.58±0.07

a,B

0.15±0.02

a,A

Tr Tr

0.67±0.08

b,B

ab,B

A

Tr

Tr

Tr

Tr

Tr

Tr

Tr

Tr

Tr

A

6.32±0.08

ab,D

0.27±0.05

6.20±0.02

A

0.58±0.04

0.12±0.03

ab,D

6.15±0.08

b,C

4.86±0.04a,B

ab,B

4.73±0.04

a,AB

a,A

4.68±0.06

a,AB a,AB

b,C

5.15±0.01 4.97±0.05

5.05±0.06

b,BC

a,A

4.80±0.04

a,AB

4.75±0.04

c,A

4.79±0.02

bc,AB

4.70±0.02

ab,A

4.60±0.03

b,A

4.67±0.03

ab,A

4.53±0.02

ab,A

0.36±0.01

b,A

0.40±0.02

ab,AB

0.65±0.02

0.47±0.02

0.68±0.04

Tr

Tr

0.13±0.03

ab,AB

a,B

0.85±0.04a,B

A

0.28±0.04

0.50±0.06

2.05±0.08

b,A

1.20±0.04

1.62±0.07

D

b,C

0.31±0.06

0.25±0.02

ab,A

0.47±0.03

3.85±0.11

a,B

Tr

ab,B

4.75±0.05

a,AB

C

Tr

b,A

4.85±0.01

ILB

3.90±0.10

ab,A

b,A

4.85±0.02

0.28±0.03

0.47±0.04

b,C

1.03±0.07

5.30±0.02

a,B

EP

60

0.32±0.07

2.36±0.11

b,B

4.91±0.03

a,A

0.14±0.02

ab,B

5.40±0.06

TE D

4

c,A

3.96±0.12

Tr ab,C

30

1

ILC D

Tr

0.02±0.01

(g Lactic

ab,AB

b,B

0.68±0.02

0.68±0.07

ab,B

0.72±0.04

0.72±0.08

ab,B

0.72±0.07

ab,B

0.54±0.04

b,BC

0.68±0.04

ab,BC

0.72±0.05

0.79±0.08

ab,C

0.79±0.08

ab,C

a,C

a,A

b,A b,B

b,BC

b,BC

0.72±0.02

ab,BC

0.92±0.05

0.83±0.06

ab,C

1.08±0.06

b,D

0.90±0.05

ab,C

1.08±0.06

b,D

b,CD

66.3±2.1D

68.7±2.0C

68.7±1.9E

68.8±1.2E

4

58.6±1.7

C

54.0±1.5

B

53.9±1.4

D

55.0±1.7

D

52.3±1.0

C

15

53.2±1.0

BC

49.8±1.7

B

49.1±1.1

CD

49.9±1.0

CD

48.7±0.9

C

30

46.8±0.9

AB

45.8±2.1

AB

44.9±1.0

BC

44.5±0.9

B

AC C

1

42.2±1.2

A

60

39.5±0.7

A

1

1.6±0.1

45

(% w/w)

0.97±0.06

a,B

Tr

1

Ash

2.16±0.08

b,C

15

Acidity

(% w/w)

3.90±0.15

Tr a,B

1

Moisture

FLB D

Tr

Glucose/Galactose

pH

D

RI PT

(g/100g cheese)

day

SC

Lactose

Whey cheese type

Maturation

M AN U

Analysis

A B B

4

7.7±0.4

15

9.1±0.4

B

30

9.9±0.4

45

10.1±0.7

B

10.3±1.0

B

60

40.5±1.0

A

38.5±0.5

A

1.5±0.2

A

7.4±0.3

B

8.7±0.5

B

9.2±0.4

B

9.8±0.7

B

9.7±0.7

B

39.7±0.8

AB

38.0±0.3 1.5±0.2

A

A

7.3±0.4

B

8.9±0.1

C

9.3±0.1

C

9.3±0.4

C

9.2±0.2

C

40.4±0.4

67.0±1.1D

BC

42.8±1.0

AB

40.2±0.6AB

A

37.3±0.3

37.6±0.2

A

A

1.5±0.2

7.3±0.2

B

7.3±0.5

B

8.6±0.4

B

8.4±0.5

B

9.4±0.5

B

9.8±0.4

B

9.3±0.5

B

9.8±0.6

B

9.2±0.4

B

9.7±0.8

B

1.5±0.1

A

ACCEPTED MANUSCRIPT

Means within a row with different lowercase superscripts differ significantly (P<0.05).

A-E

Means within a column

with different uppercase superscripts in the same analysis differ significantly (P<0.05). C: control whey cheese produced without culture, FLC: whey cheese produced with free L. casei ATCC 393 cells, FLB: whey cheese produced with free L. bulgaricus ATCC 11842 cells, ILC: whey cheese produced with immobilized L. casei ATCC

EP

TE D

M AN U

SC

RI PT

393 cells on casein, ILB: whey cheese produced with immobilized L. bulgaricus ATCC 11842 cells on casein.

AC C

a-d

ACCEPTED MANUSCRIPT

Table 4. Effect of different lactobacilli cultures on major aroma-related compounds (μg/kg) of novel dried whey cheeses after ripening for 60 days. Identification

Compound

C

FLC

FLB

ILC

ILB

Esters RT, KI, MS

Nd

Nd

Ehtyl hexanoate

RT, KI, MS

2.1

0.3

Ethyl octanoate

RT, KI, MS

8.3

4.4

Ethyl decanoate

RT, KI, MS

15.0

Ethyl dodecanoate

KI, MS

8.5

Ethyl tetradecanoate

KI, MS

Nd

Ethyl hexadecanoate

KI, MS

Nd

3-Methylbutyl acetate

KI, MS

Nd

Methyl decanoate 2-Phenylethyl acetate Ethyl-9-hexadecenoate Organic acids Pentanoic acid Octanoic acid Nonanoic acid Decanoic acid

Nd

Nd

1.1

2.2

5.1

5.2

Nd

19.9

14.8

16.5

34.6

7.9

Nd

5.6

11.2

14.0

Nd

6.6

Nd

12.6

Nd

4.5

7.8

Nd

Nd

Nd

3.7

6.2

Nd

1.0

Nd

2.1

MS

4.5

Nd

Nd

Nd

Nd

RT, KI, MS

9.8

3.5

6.8

5.4

2.8

KI, MS

Nd

16.5

Nd

8.2

11.6

MS

Nd

Nd

Nd

Nd

11.0

KI, MS

Nd

29.6

17.4

4.3

22.0

RT, KI, MS

RT, KI, MS RT, KI, MS

118.1 44.7 114.2 62.3 197.8 Nd

15.5

Nd

7.3

23.7

286.3 350.9 662.8 397.8 566.2

KI, MS

80.4

52.9

43.8

71.6 104.3

2-Methyl butanoic acid

KI, MS

Nd

31.6

Nd

Nd

36.2

3-Methyl butanoic acid

MS

Nd

Nd

Nd

Nd

33.8

2-Ethyl hexanoic acid

MS

Nd

2.8

Nd

Nd

Nd

10-Undecenoic acid

KI, MS

Nd

Nd

Nd

Nd

1.5

AC C

EP

Dodecanoic acid

KI, MS

TE D

Hexanoic acid

0.3

M AN U

3-Methylbutyl hexanoate

Nd

SC

Ethyl butanoate

RI PT

method

Alcohols

1-Hexanol

RT, KI, MS

Nd

Nd

0.8

Nd

Nd

1-Heptanol

KI, MS

Nd

Nd

1.3

Nd

Nd

1-Octanol

RT, KI, MS

Nd

Nd

1.0

1.5

Nd

1-Nonanol

RT, KI, MS

Nd

Nd

Nd

2.4

4.4

1-Dodecanol

RT, KI, MS

Nd

1.5

Nd

Nd

Nd

4-Methyl-2-pentanol

RT, KI, MS

11.6

0.3

Nd

Nd

Nd

3-Methyl-1-butanol

RT, KI, MS

12.0

15

10.5

Nd

Nd

2-Heptanol

RT, KI, MS

9.0

4.2

4.9

1.9

Nd

2-Nonanol

RT, KI, MS

7.6

5.5

10.0

4.9

Nd

ACCEPTED MANUSCRIPT

RT, KI, MS

10.9

Nd

Nd

Nd

Nd

2-Ethyl-1-hexanol

RT, KI, MS

7.1

7.6

2.8

1.6

Nd

1,3-Butanediol

KI, MS

Nd

Nd

Nd

Nd

51.8

Phenyl ethanol

RT, KI, MS

56.4

95.2

16.5

59.8

74.7

Phenol

RT, KI, MS

5.6

Nd

3.4

2.7

8.5

3- Methyl-phenol (m-cresol)

MS

Nd

Nd

7.4

21.2

2.7

4-Methyl-phenol (p-cresol)

KI, MS

2.3

13.6

8.5

46.9

8.6

3-Methyl butanal

KI, MS

Nd

Nd

Nd

43.2

Nd

2-Heptanone

KI, MS

26.6

43.1

36.5

11.7

5.4

2-Octanone

MS

4.2

1.2

1.1

0.3

0.6

KI, MS

7.9

2-Pentadecanone 3-Hydroxy-2-butanone (acetoin) Benzaldehyde Lactones γ-Decalactone δ-Decalactone γ-Dodecalactone δ-Dodecalactone

MS

Nd

KI, MS

Nd

4.9

22.4

7.9

15.4

Nd

Nd

2.5

Nd

20.5

0.9

3.1

27.9

M AN U

2-Undecanone

SC

Carbonyl compounds

RI PT

1-Octen-3-ol

KI,MS

27.5

8.9

4.6

10.5

18.9

KI, MS

Nd

4.5

Nd

Nd

Nd

KI, MS

Nd

8.1

2.5

Nd

Nd

KI, MS

8.2

15.4

6.8

24.3

14.4

KI, MS

2.9

2.9

Nd

3.7

2.8

TE D

RT: Positive identification by retention times that agree with authentic compounds generated in the laboratory, KI: Tentative identification by kovats’ retention index compared to the literature, MS: tentative identification by mass spectra obtained from NIST107, NIST21 and SZTERP libraries, C: control whey cheese produced without culture, FLC: whey cheese produced with free L. casei ATCC 393 cells, FLB: whey cheese produced with free L. bulgaricus ATCC 11842 cells, ILC: whey cheese produced with immobilized L. casei ATCC 393 cells on casein,

AC C

EP

ILB: whey cheese produced with immobilized L. bulgaricus ATCC 11842 cells on casein, Nd: non detected.

ACCEPTED MANUSCRIPT

Table 5. Effect of different lactobacilli cultures on major aroma-related groups (μg/kg of cheese) of novel dried whey cheeses after ripening for 60 days. C

Compound

FLC

FLB

ILC

ILB

μg/kg ab

c

a

ab

c

54.4±2.5 81.2±3.6 26.7±1.9 54.4±3.1 73.9±3.9 a a b a c 484.8±13.8 528.0±16.2 838.2±13.9 543.3±10.8 996.5±23.7 b b a b b 122.5±5.7 142.9±4.4 67.1±2.7 142.9±6.9 150.7±7.8 ab ab a b ab 66.2±2.3 78.6±3.1 65.5±1.9 79.2±2.7 68.2±1.4 a b a b a 11.1±1.9 30.9±2.0 9.3±0.9 28.0±1.7 17.2±1.3

Total

739.0±23.8a 861.6±19.9ab 1006.8±29.8b 847.8±20.3a 1306.5±30.7c

RI PT

Esters Organic acids Alcohols Carbonyl compounds Lactones a-c

SC

Means within a row with different lowercase superscripts differ significantly (P<0.05). C: control whey cheese produced without culture, FLC: whey cheese produced with free L. casei ATCC 393 cells, FLB: whey cheese

M AN U

produced with free L. bulgaricus ATCC 11842 cells, ILC: whey cheese produced with immobilized L. casei ATCC

AC C

EP

TE D

393 cells on casein, ILB: whey cheese produced with immobilized L. bulgaricus ATCC 11842 cells on casein.

ACCEPTED MANUSCRIPT

Table 2. Effect of different lactobacilli cultures on total aerobic, lactococci and lactobacilli counts (log cfu/g) of novel dried whey cheeses during ripening. Whey cheese type

Maturation C

Total aerobic

1

5.5±0.2

count

4

6.7±0.2

a,AB BC

FLB 6.8±0.2

ab,AB

7.9±0.4

7.0±0.3 7.8±0.2

8.4±0.4

AB

8.5±0.3

B

B

9.5±0.2

B

B

9.5±0.5

B

A

5.4±0.1

AB

6.5±0.2

8.7±0.4

C

8.8±0.5

45

9.0±0.5

C

9.2±0.4

60

9.1±0.4

C

9.4±0.4

Mesophilic

1

5.4±0.1

A

5.3±0.2

lactococci

4

6.5±0.3

AB

6.3±0.3

15

7.8±0.2BC

30

8.3±0.4

C

45

8.3±0.3

C

60

8.3±0.4

C

Thermophilic

1

4.8±0.1

lactococci

4

6.0±0.3

15

7.7±0.2

30

7.8±0.3B

Lactobacilli

1 4

8.0±0.4

B

8.2±0.3

B

4.0±0.1

a,A

5.9±0.3

a,B

7.2±0.3

B

30

8.8±0.4

45 60

AC C

AB

6.7±0.1

b,AB

8.3±0.2

7.2±0.4

8.3±0.2

B

9.3±0.4

B

9.3±0.4

B

9.7±0.2

B

9.9±0.4

B

9.8±0.3

B

9.9±0.5

B

5.7±0.2

AB

6.7±0.3

M AN U

A

5.8±0.2

AB

6.8±0.2

7.8±0.3BC

C

8.2±0.3

C

8.5±0.2

C

8.7±0.3

C

7.8±0.3

BC

7.9±0.3

7.8±0.2

ab,A

5.1±0.2

5.1±0.2

ab,A

B

7.1±0.2

8.0±0.3

B

8.2±0.2

8.2±0.4B

8.3±0.3BC

6.9±0.2

B

8.4±0.3

8.2±0.3

b,A

6.4±0.2

8.0±0.3

b,AB

7.7±0.1

8.3±0.3

C

8.9±0.3

8.9±0.3

C

9.4±0.4

8.9±0.3

C

9.3±0.4

7.9±0.2

5.6±0.1

C

8.5±0.3

C

BC

7.7±0.2

ab,A

5.9±0.1

8.3±0.2

8.4±0.3

b,A

7.2±0.3

b,A

7.2±0.2

BC

8.3±0.2

8.9±0.3

BC

C

b,A

6.9±0.2

8.3±0.3

8.7±0.3

BC

8.8±0.3

B

C

9.4±0.4

BC

9.2±0.4

BC

8.9±0.3 8.2±0.2

b,AB

B

8.9±0.3C

BC

B

BC

B

8.9±0.3C

8.0±0.2

7.8±0.3BC C

BC

b,AB

AB

8.4±0.4

7.2±0.3

C

A

BC

B

8.7±0.4

B

b,AB

8.9±0.2

8.8±0.5

A

7.5±0.3BC

ab,A

AB

8.2±0.4

8.5±0.3

6.2±0.1

ILB b,A

C

8.0±0.2

BC

EP

15

7.5±0.3BC

TE D

60

B

ab,AB

8.3±0.3

30

A

ab,A

AB

7.7±0.3

a,A

ILC

b,A

15

45

a-b

FLC a,A

RI PT

day

SC

Microorganism

8.2±0.3

C

BC

b,A

b,AB

AB

8.8±0.3B

9.5±0.3

B

9.4±0.4

B

B

9.7±0.4

B

9.8±0.4

B

B

9.6±0.4

B

9.7±0.4

B

Means within a row with different lowercase superscripts differ significantly (P<0.05).

A-C

Means within a column

with different uppercase superscripts in the same analysis differ significantly (P<0.05). C: control whey cheese produced without culture, FLC: whey cheese produced with free L. casei ATCC 393 cells, FLB: whey cheese produced with free L. bulgaricus ATCC 11842 cells, ILC: whey cheese produced with immobilized L. casei ATCC 393 cells on casein, ILB: whey cheese produced with immobilized L. bulgaricus ATCC 11842 cells on casein.

ACCEPTED MANUSCRIPT

Table 3. Effect of different lactobacilli cultures on yeasts, coliforms, enterobacteria and staphylococci counts (log cfu/g) of novel dried whey cheeses during ripening.

C

1

5.3±0.2

4

5.4±0.2

AB

15

Enterobacteria

b,AB

5.2±0.3

5.3±0.3

5.2±0.3

C

5.1±0.2

B

4.5±0.2

4.7±0.3

a,B

a,ABC

a,AB

4.1±0.2

a,A

3.4±0.1

a,A

a,A

3.3±0.1

a,A

45

5.0±0.2

b,A

3.4±0.1

60

4.9±0.2

b,A

3.1±0.1

1

4.7±0.1

A

4.6±0.2

BC

4.7±0.2

4

5.4±0.1

AB

4.9±0.3

BC

5.0±0.4

15

6.2±0.3b,BC

30

6.5±0.4

b,C

b,ABC

45

5.9±0.2

60

5.1±0.2

b,AB A

1

4.7±0.1

4

5.5±0.2

15

6.1±0.3

30

6.5±0.4b,B

1

AB

b,AB

4.0±0.2

a,BC

5.8±0.3

60

5.6±0.3ab,C

a,AB

4.0±0.2

4.0±0.1

a,AB

5.2±0.3

C

5.3±0.3

C

4.4±0.2

4.1±0.2

a,BC

4.0±0.2

a,AB

3.2±0.1

a,A

2.8±0.1

3.3±0.1

3.0±0.1

C

B

4.6±0.3

B

4.7±0.2

C

4.5±0.2a,B

a,A

2.9±0.1

4.8±0.2

BC

4.6±0.2

BC

4.8±0.2

4.9±0.2

BC

5.2±0.3

BC

4.6±0.2

4.2±0.2a,AB

5.1±0.2

b,AB

3.2±0.1

A

4.2±0.2

4.0±0.1

a,AB

ab,C

4.2±0.2a,AB 3.8±0.1

a,AB

a,A

3.3±0.1

ABC

4.3±0.2

5.8±0.3

5.3±0.3

30

6.3±0.4

b,B

4.8±0.2

45

5.7±0.3

b,B

3.8±0.2

60

5.3±0.2

b,AB

3.0±0.1

5.0±0.3

5.3±0.2

ab,BC

a,A

4.7±0.2

3.3±0.1

ab,C

a,AB

B

a,A

5.4±0.3

a,ABC

4.6±0.3

3.8±0.2

3.3±0.1

4.4±0.2

a,BC

a,ABC

a,A

b,AB

EP

C

3.5±0.2

b,B

AC C

5.4±0.3

4.4±0.2

5.7±0.3

15

5.2±0.3

AB

5.5±0.2

4.3±0.2

ILB C

AB

4.7±0.3ab,AB

4.1±0.2

b,B

4

a-b

ILC B

30

45

Staphylococci

6.5±0.3

b,B

FLB C

TE D

Coliforms

FLC AB

RI PT

day

SC

Yeasts/Molds

Whey cheese type

Maturation

M AN U

Microorganism

a,A

ABC

a,B

4.4±0.2

a,AB

3.7±0.1

a,A

2.9±0.1

4.9±0.1

a,BC

a,AB a,A

C

4.6±0.2

C

BC

4.5±0.2

C

ab,C

4.5±0.2a,BC 3.8±0.1

4.7±0.2a,C

4.7±0.1

4.3±0.2a,BC

a,AB

3.5±0.2

a,A

2.8±0.1

3.0±0.1

4.0±0.3

B

a,C

a,AB a,A

4.1±0.2

C

4.7±0.2

ab,BC

4.2±0.2

a,B

ab,C

5.3±0.3

ab,BC

4.2±0.2

a,B

4.2±0.2a,C

a,BC

4.7±0.2

a,BC

4.2±0.2

a,B

4.1±0.1

a,AB

3.7±0.2

a,AB

3.1±0.1

a,AB

3.2±0.2

a,A

3.0±0.1

a,A

2.7±0.1

a,A

2.5±0.1

Means within a row with different lowercase superscripts differ significantly (P<0.05).

4.2±0.2

a,C

a,BC

a,AB a,A

A-C

Means within a column

with different uppercase superscripts in the same analysis differ significantly (P<0.05). C: control whey cheese produced without culture, FLC: whey cheese produced with free L. casei ATCC 393 cells, FLB: whey cheese produced with free L. bulgaricus ATCC 11842 cells, ILC: whey cheese produced with immobilized L. casei ATCC 393 cells on casein, ILB: whey cheese produced with immobilized L. bulgaricus ATCC 11842 cells on casein.

ACCEPTED MANUSCRIPT

Research highlights

Novel whey cheese production by thermally dried lactobacilli

•

Casein was used as immobilization carrier

•

Novel whey cheeses with improved quality and extended shelf life were produced

•

Counts of coliforms, enterobacteria and staphylococci were significantly reduced

AC C

EP

TE D

M AN U

SC

RI PT

•