Effect of free and microencapsulated Lactococcus lactis on composition and rheological properties of Manchego-type cheeses during ripening

Accepted Manuscript Effect of free and microencapsulated Lactococcus lactis on composition and rheological properties of Manchego-type cheeses during ripening

Juan Alfredo Salazar-Montoya, Rafael González-Cuello, Emmanuel Flores-Girón, Emma Gloria Ramos-Ramírez PII: DOI: Reference:

S0963-9969(17)30752-4 doi:10.1016/j.foodres.2017.10.067 FRIN 7115

To appear in:

Food Research International

Received date: Revised date: Accepted date:

16 August 2017 26 October 2017 28 October 2017

Please cite this article as: Juan Alfredo Salazar-Montoya, Rafael González-Cuello, Emmanuel Flores-Girón, Emma Gloria Ramos-Ramírez , Effect of free and microencapsulated Lactococcus lactis on composition and rheological properties of Manchego-type cheeses during ripening. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/ j.foodres.2017.10.067

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ACCEPTED MANUSCRIPT

Effect of free and microencapsulated Lactococcus lactis on composition and rheological properties of Manchego-type

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cheeses during ripening.

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Juan Alfredo Salazar-Montoya*, Rafael González-Cuello, Emmanuel Flores-Girón, Emma Gloria Ramos-Ramírez.

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* Corresponding author:

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Biotechnology and Bioengineering Department. CINVESTAV-IPN Av. IPN 2508. Col. San Pedro Zacatenco. México City. Mexico.

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 Juan Alfredo Salazar-Montoya Email address: [email protected]

CINVESTAV-IPN (Center for Research and Advanced Studies. National Polytechnic Institute) CINVESTAV-IPN. Biotechnology and Bioengineering Department. Av. IPN 2508. Col. San Pedro Zacatenco. México City. Mexico. Tel: 52(55)57473800 ext. 4364

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ABSTRACT

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This study aimed at determining the composition and rheology changes during the ripening

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(60 days) of Manchego-type cheeses prepared with Lactococcus lactis subsp lactis and

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Lactococcus lactis subsp cremoris in the free status and microencapsulated in gellan gum. The composition indicated that microencapsulation had a greater influence than the period

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of ripening since discrepancies were statistically significant (p<0.05) regarding the levels

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of protein, moisture, total solids and pH between the two cheeses both at the beginning and end of the ripening. Both cheeses presented predominantly elastic characteristics (Gʼ>G”).

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The moduli and the viscosity significantly increased with the presence of microcapsules

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and, ripening time. Conversely, the recovery percentages on the creep curve decreased in both cheeses with an increase in ripening as a result of the degradation of proteins. The

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retarded time ranged between 2.32x10-2 and 2.38x10-2 s and the Jo values were 29 times

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higher than the Ji values in the studied cheeses, indicating the loss of elasticity of the

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cheeses as they ripen.

Keywords: Rheological behavior, Microencapsulation, Lactococcus lactis, Manchegotype cheese, Gellan gum, viscoelasticity.

ACCEPTED MANUSCRIPT 1. Introduction Manchego-type cheese is one of the most economically important ripened cheeses in Mexico, with an output of 32,630 tons per year (INEGI, 2017). It is a product made from pasteurized whole cow's milk and calf rennet extract, unlike original Manchego cheese in Spain (Lobato-Calleros, Velázquez-Varela, Sánchez-

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prepared from sheep milk

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García, & Vernon-Carter, 2003) and it is a semi-hard pressed cheese subjected to

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maturing processes for a minimum of 7 days at a controlled temperature and moisture (NMX-F-462-1984). The maturing processes or cheese ripening is a long and expensive

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process due to the immobile capital, weight loss, cold storage and fermentation caused by

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undesirable microorganisms but indispensable since in this process the lactic acid bacteria (LAB) conduct to the production of the majority of the sensory attributes and

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unique characteristics of the cheeses (Gaglio, et al., 2014; Guarcello et al., 2016). Thus,

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shortening the ripening period can lead to a considerable reduction in the production costs of cheese (Garde, Tomillo, Gaya, Medina, & Muñez, 2002). Recently, microencapsulation

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of LAB with functional biopolymers has been used to accelerate the ripening process of

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cheeses (Özer, Kirmaci, Senel, Atamer, & Hayaloǧlu, 2009). Microencapsulation is a process by which bioactive substances are retained within a

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matrix or wall system in order to protect them from deleterious environmental conditions, thus preventing losses and allowing for the gradual release of the substances under controlled conditions (Anal, Stevens, & Remuñan, 2006; Yáñez, Salazar, Chaires, Jiménez, Márquez, & Ramos, 2002). During the ripening period, microcapsules can avoid the loss of enzymes (Anjani, Kailasapathy, & Phillips, 2007) and microorganisms, which substantially improves the efficiency of the process. For example, microencapsulation has been

ACCEPTED MANUSCRIPT recommended to protect bacteria used in cheese production from adverse environmental conditions that occur inside these products (Karimi, Mortazavian, & Gomes, 2010). According to Özer, Kirmaci, Senel, Atamer, and Hayaloǧlu (2009), the microencapsulation does not adversely affect the appearance of experimental cheeses. However, further studies

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are needed to evaluate the quality of the obtained products.

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One way to evaluate food quality as a function of storage conditions is through

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rheological characterization (Karoui, Laguet, & Dufor, 2003), which is correlated with the texture, sensory attributes, composition and microstructural changes that occur in food

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products (Shoemaker, Nantz, Bonnans, & Noble, 1992). Cheese is a typically viscoelastic

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food, whose rheological properties are largely influenced by its chemical composition, i.e., total solids, fat, salts, and pH (Jaros, Petrag, Rohm, & Ulberth, 2001). This influence is

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largely due to the stages of proteolysis (Lawrence, Gilles, & Creamer, 1999) and lipolysis

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that take place during the cheese ripening process (Bachmann, Bütikofer, & Meyer, 1999). Nowadays, no investigations have been conducted pertaining to the composition and behavior

of

Manchego-type

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rheological

cheeses

made

with

microencapsulated

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microorganisms. Therefore, this type of information could be useful. for the purpose of comparing the microencapsulation technology with the "traditionally" manufactured

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Manchego-type cheese, whereas the new microstructure that could be formed in the cheese matrix could be used as a quality control tool. The objective of this study was the evaluation of the composition and rheological properties of Manchego-type cheeses obtained with and without microencapsulated microorganisms. 2. Materials and methods 2.1. Microencapsulation 2.1.1. Dispersion preparation

ACCEPTED MANUSCRIPT Gellan gum (Kelco Biopolymers. Monsanto, USA) dispersions were prepared with deionized water at a 0.2% concentration using a blend of high (HA) and low acylated (LA) gellan (25 HA:75 LA) (González-Cuello, Ramos-Ramírez, & Salazar-Montoya, 2010). Subsequently, calcium was added (30 mM) (Huang, Tang, Swanson, & Rosco, 2003) and

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dispersed by constant stirring at 90 °C for 10 min on a hot plate stirrer (model 502C,

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Barnstead Thermolyne, USA).

2.1.2. Emulsion preparation

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Dispersion of biopolymers with HA and LA gellan gum were mixed with a blend of

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Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris at concentration of 8.5 log CFU/ml, the counting was carried out in culture dishes with M17 agar (see 2.3). The

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emulsions were then prepared via the addition of 0.2% Span 80 (sorbitan monooleate)

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supplied by Sigma-Aldrich (USA) in vegetable oil under constant stirring at 800 rpm on a hot plate stirrer (model 502C, Barnstead Thermolyne, USA); these parameters are

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appropriate to obtain microcapsules with sizes between 15-75 µm (González-Cuello,

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Ramos-Ramírez, & Salazar-Montoya, 2010), which represent adequate diameters for food applications (Tyle, 1993). Then, α-gluconolactone (Sigma-Aldrich, USA) was added until a

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pH of 4.5 was reached to start the gelation process. Finally, the oil was removed by adsorption, and the microcapsules contained in the aqueous phase were centrifuged twice at 5000 rpm for 10 min (Universal 30RF Hettich-Zentrifugen, Germany) with saline solution and stored at 4 °C until use.

2.2. Manchego-type cheese

ACCEPTED MANUSCRIPT Cheeses made with the microorganisms in the free status (CFM) and cheeses made with microencapsulated microorganisms (CMM) were prepared according to the traditional process. The whole milk was purchased at a local supermarket supermarket which was then vat-pasteurized at 63C for 30 min, 1.5 mL/L of a calcium chloride solution

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10%(w/w) was added to pasteurized milk at 30C. The lactic culture (Choozit ma 25

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11lyo DCU) was supplied by Danisco (México), and the rennet used was of pharmaceutical

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degree (Strength 1:7500. Qualact, Altecsa S.A de C.V, México) coagulation process

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occurred at 30±1C. Both cheeses (CFM and CMM) were done by triplicate. 2.3. Determination of Lactococcus lactis

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For the quantification of free and microencapsulated microorganisms in Manchego-type cheeses, consecutive dilutions at 10-5 from a sample of 10 g of cheese were made in

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peptone water (Casein peptone, BD Bioxon. México). Later, the counting was carried out

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in culture dishes with M17 agar (DifcoTM. Becton, Dickinson and Company Sparks, USA)

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agar at 35 °C ± 0.2 after 48 h of incubation, for each experimental unit the analysis was realized by triplicate. The release of the microencapsulated microorganisms was carried

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out by the technique used by Yañez (2007) which implicated a mechanical breakup of

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microcapsules by stirring.

2.4. Compositional analysis of cheese During the ripening period (a total of 60 days) of Manchego-type cheeses, the following bromatological parameters were determined: fat, protein, moisture, total solids, ash, pH, and chlorides, as specified in the Mexican standard NMX-F-462 (1984).

ACCEPTED MANUSCRIPT 2.5. Dynamic rheology The determinations were performed in a LS 100 low stress rheometer (Paar Physica, Germany). A parallel plate (PP20) geometry was employed with a diameter of 20 mm and a 3 mm gap. The samples were placed in the bottom plate (Peltier), and the gap was set.

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Measurements were conducted at shear rates between 5.00x10-5 and 1.00x103 s-1 and shear

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stresses between 6.37x10-1 and 6.37x103 Pa. The data analysis was realized with the

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2.5.1. Linear viscoelastic region

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equipment software Paar Physica ver. 206-E.

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The studies were conducted with a sweep of the amplitude with torques between 1 x 10-3 and 1 x 101 mNm to determine the linear viscoelastic region, which was obtained when Gʼ

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and G” moduli were independent of the amplitude; subsequently, a frequency sweep was

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performed between 0.010 and 0.50 Hz in order to determine the behavior of viscous and

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elastic components versus the frequency.

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2.5.2. Creep recovery curves

This test consisted of applying an instantaneous stress, which was kept constant, and

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monitoring the deformation of the material as a function of time (Augusto, Ibarz, & Cristianini, 2013; Jiménez-Avalos, Ramos-Ramírez, & Salazar-Montoya, 2005; Silva et al., 2017). The results were in compliance with terms adjusted to the Burger´s model [Eq. 1]. The software Paar Physica ver. 2.06-E was used to calculate the following dynamic parameters: instantaneous compliance (Jo, Pa-1), retarded compliance (JR, Pa-1), relaxation time (λret, s), experimental time (t), and viscosity at zero shear rate (ηN).

ACCEPTED MANUSCRIPT    t  t   J(t)  J0  JR 1 exp  ret  N 

(1)

2.6. Statistical analysis Differences between mean values of the composition and rheological analysis were

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determined by ANOVA (one way) using the test LSD (least significance difference) at a

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significance level of =0.05 through the software SPSS ver. 13.0 (SPSS Inc. USA). Each

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treatment was done by triplicate in a completely randomized design.

3. Results and discussion

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3.1. Determination of Lactococcus lactis

There was a significant increase in the population of Lactococcus lactis in the studied

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Manchego-type cheeses (CFM and CMM)during ripening (Table 1). This behavior can be

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correlated with the decrease in the pH of the cheeses, which indicates the metabolic activity of microorganisms in Manchego-type cheeses (CFM and CMM). In both cheeses,

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microorganisms remained viable throughout the ripening process (Bergamini, Hynes,

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Quiberoni, Suárez, & Zalazar, 2005). The population of Lactococcus lactis in CFM samples presented statistical similarity (p

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>0.05) from day 30 (8.57 log10CFUg-1) to day 60 (8.51 log10CFUg-1) without a decrease in the microorganism population at the end of the ripening time. The results of this study are similar to those reported by Cabezas, Sánchez, Poveda, Seseña, and Palop (2007) in Manchego cheeses within sixty days of ripening, where the amounts of Lactococcus spp without microencapsulation were between 8.12 and 8.54 log10CFUg-1 in Manchego cheeses from two different procedures. On the other hand, the CMM presented average values that

ACCEPTED MANUSCRIPT were 1.17 times greater than those of the CFM, exhibiting statistical difference (p<0.05) throughout the entire ripening period. In the case of CMM cheeses, significant differences were found at the beginning of ripening (first day) (8.32 log10CFUg-1) until 60 days of ripening (9.96 log10CFUg-1), which indicates the great potential of microencapsulation as a

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preservation technique for microorganisms in food systems.

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3.2. Composition of Manchego-type cheeses

The compositional evolution of Manchego-type cheeses is presented in Table 2, where

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differences in the composition suggested biochemical changes during ripening. We

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observed a decrease in the fat content in CFM and CMM cheeses as a result of the metabolism of the microorganisms used. However, the cheeses do not show a sudden drop

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in the fat content, presumably by the ripening time (60 days). In CFM, no statistically

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significant differences were observed (p >0.05) during the first 30 days of ripening, whereas in CMM, changes occur after 15 days of ripening. The lowest concentration of fat

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was presented at 60 days of ripening for CMM (26.33%), with statistical differences

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compared to the other studied Manchego-type cheeses. The determination of fat is important because its hydrolysis has an important role in the formation of aromas (Fox,

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Law, McSweeney, & Wallace, 1993). Our results regarding the fat levels were lower than those reported by Cabezas, Sánchez, Poveda, Seseña, and Palop (2007) (53.11 to 56.36%) in Manchego cheeses; this difference is caused by the type of raw material used and the kind of microorganisms that carry out the ripening. However, our results at the beginning of ripening were similar to those reported

ACCEPTED MANUSCRIPT by Lobato-Calleros, Velázquez-Varela, Sánchez-García, and Vernon-Carter (2003) (30.5%) for Manchego-type cheeses with microorganisms in the free status. By applying ANOVA statistical analysis (one way) to the obtained pH values, statistical differences (p<0.05) were established among all of the ripening period in both of the

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studied treatments due to the production of lactic acid by Lactococcus lactis. This decrease

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in pH affects the moisture, as long as the cheese syneresis increases (van Vliet & Walstra,

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1994), represented by the structural reorganization induced by the protein-protein bonds in the casein gels. On the other hand, some authors (Cabezas, Sánchez, Poveda, Seseña, &

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Palop, 2007) have reported that the pH of cheeses increases during ripening as a result of

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the consumption of lactic acid and the alkaline effect of compounds generated during protein degradation.

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It is recognized that changes occur during ripening in cheese (Gunasekeran & Ark,

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2002) where proteolysis is responsible for textural changes (Fox, Guinee, Cogan, & McSweeney, 2000). Therefore, it is important to study the behavior of proteins during

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ripening.

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During CFM ripening, no significant decreases were observed (p >0.05) in protein concentration during the first 30 days of ripening, which reveals a slow degradation of

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casein by microorganisms in the free status. On the other hand, in CMM, there were significant differences (p<0.05) after 15 days of ripening. The highest concentration of protein of 25.96% was obtained at the beginning of ripening in the CMM; this could indicate a possible interaction between the whey proteins and gellan gum (Sanderson, & Clark, 1983). Paradoxically, lower concentrations of protein are also found in the CMM at the end of ripening (22.52%), and this implies a higher decrease in protein levels in CMM

ACCEPTED MANUSCRIPT than in CFM. Overall, the results found in this study in the CFM were similar to those reported by Lobato-Calleros, Velázquez-Varela, Sánchez-García, & Vernon-Carter (2003), who found a protein concentration of 24.6% in fresh Manchego-type cheeses. The decrease in protein levels is caused mainly by the activity of microorganisms since

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the degradation of casein by rennet enzymes decreases at pH values below 5.8 (Fox, Law,

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McSweeney, & Wallace, 1993), even though one must not rule out possible cell lysis,

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which leads to the release of intracellular peptidases into the cheese (Hynes, Bergamini, Suárez, & Zalazar, 2003). Lactococcus lactis has a well-characterized proteolytic system

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including proteinases adhered to the cell wall capable of hydrolyzing casein into small

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peptides and free amino acids (Yvon & Rijnen, 2001). Therefore, cell lysis of lactic cultures is proposed in cheeses as a way to speed up biochemical reactions involved in

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flavor reactions (Crow, Coolbear, Gopal, Martley, McKay, & Riepe, 1995), with this being

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a possible explanation for the rapid decline of protein in the CMM compared with the CFM since the microcapsules could cause rupture of the membrane of Lactococcus lactis.

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However, Kailasapathy & Masondole (2005) had reported a possible microcapsule

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degradation by the effect of sodium chloride and the subsequent microbial release inside the cheese. Proteolysis in cheeses must be studied in more detail because it can cause

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defects in taste and texture in the final product (Beresford & Williams, 2004). Though, further investigation needs to be performed in order to corroborate these hypotheses. The moisture content decreases in both cheeses at 15 days of ripening as a result of the environmental conditions that they were exposed to (80% RH and 10 °C). However, the moisture values obtained in both Manchego-type cheeses were greater than those reported by Lobato-Calleros, Velázquez-Varela, Sánchez-García, and Vernon-Carter (2003) (38.4%)

ACCEPTED MANUSCRIPT in fresh Manchego-type cheeses, and this may reflect differences in the methodologies used in cheese manufacture. After 15 days of ripening, the moisture gradually decreases as the cheese matures as a result of syneresis, which occurs by the rearrangement of the protein network producing large amounts of the expulsion of whey (Lobato-Calleros, Aguirre-

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Mandujano, Vernon-Carter, & Sánchez-García, 2000). The moisture content of the CMM

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was significantly higher (p<0.05) than that of CFM due to the hydrophilic nature of the

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gum used (gellan), which retains moisture in the cheese (Kanombirira & Kailasapathy, 1995).

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With respect to the total solids, CMM had the highest concentrations (54.54 to 55.84%)

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as a result of the presence of gellan gum and calcium carbonate in the microcapsules. In conclusion, the total solids increase throughout the ripening process as a result of the

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above-mentioned syneresis. Similar concentrations in Manchego-type cheeses were

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reported by Cabezas, Sánchez, Poveda, Seseña, and Palop (2007), which showed values of

60% at 60 days.

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solid total solids between 57.54 and 54.95% at the beginning of ripening and approximately

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The ash concentrations were higher in the CMM (1.26 times) than those obtained in CFM; this difference is again attributed to the presence of the gellan gum and calcium

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carbonate used in the preparation of the microcapsules. In the CMM, the ash values were practically constant after 15 days of ripening, i.e., there were no statistical differences (p >0.05). To determine the quality of the cheeses, it is also necessary to quantify the chloride concentration because it affects the water activity, enzymatic activity and microbial growth, while it also contributes to cheese flavour development. The statistical differences found

ACCEPTED MANUSCRIPT in the levels of chlorides may be attributed to the movement of salt through the cheese (Mocquot, 1979) and the loss of moisture. The results obtained in this study are similar to those reported by Cabezas, Sánchez, Poveda, Seseña, and Palop (2007) in Manchego cheeses, where the chloride concentration increases slightly with the augmentation of the

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ripening time.

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After 30 days of ripening, the CMM had very similar levels of fat, protein, total solids

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and chlorides to those found in CFM at 45 days of ripening, and thus, the microbial microencapsulation could be considered as an alternative in the ripening of cheeses.

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Therefore, it is desirable to perform sensory analysis to determine the effect of

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microencapsulation on the acceptability of the product by the consumer.

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3.3. Dynamic rheological analysis of Manchego-type cheese

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Changes in the viscoelastic properties, storage and loss moduli (Gʼ and G”), and phase angle (tan δ) are presented in Table 3, where statistical differences (p<0.05) of the moduli

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with respect to ripening were obvious (Rosemberg, Wang, Chuang, & Schoemaker, 1995).

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This increase is associated with the denser and more interwoven structure of the cheese (Xiong & Kinsella, 1991). A greater elastic than viscous behavior (Gʼ>G”) (Figure 1a and

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Figure 1b) indicated the dominant contribution of the storage modulus in the viscoelasticity of the Manchego-type cheeses studied in the frequency range of 0.01 to 0.5 Hz. This represents a typical behavior of viscoelastic solids, which was also reported in cheeses by several authors (Osorio, Ciro, & Mejía, 2005; Piska, & Ŝtêtina, 2004). The increase in Gʼ and G” with ripening time in both Manchego-type cheeses (CFM and CMM) is consistent with previous studies in Cheddar cheeses (Venugopal &

ACCEPTED MANUSCRIPT Muthukumarappan, 2003). This increase may be caused by moisture loss (Muliawan & Hatzikiriakos, 2007) and by increasing the contact area between the casein particles, which in turn, is caused by increased casein-casein interactions (hydrogen bonds and electrostatic interactions) (Lucey, Johnson, & Horne, 2003). Rosenberg, Wang, Chuang, and Shoemaker

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(1995) studied the changes in the viscoelastic properties of Cheddar cheese during ripening

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and found that increases in Gʼ with ripening time are also related to the decreased

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concentration of water available for the solvation of the chains of protein. The values of tan δ are useful indices of viscoelastic materials; values <1 indicate a gel-

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like behavior (Mounsey & O'Riordan, 1999). The values found in this study are within the

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ranges of 0.264-0.342 and 0.299-0.341 for CFM and CMM, respectively. Similar results, were reported by Del Nobile, Chillo, Falcone, Laverse, Pati, and Baiano (2007), who found

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values of tan δ in Canestrello Pugliese cheese from 0.30 to 0.40. In concordance with the

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findings of Visser (1991), our results show that the composition can affect the viscoelastic behavior of cheeses.

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3.4. Creep and recovery curves

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Figure 2 shows the creep and recovery curves of CFM (Fig. 2a) and CMM (Fig. 2b). In all of the studied cheeses, recovery appeared when the applied stress was suspended. The

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observed recoveries were between 70.67 and 48.63% for CFM and between 70.97 and 45.24% for CFM; i.e., as long as the ripening time of the cheese was higher, the recovery percentage decreased. The behavior presented by the cheeses is that of a viscoelastic solid. These results are in accordance to those reported by Osorio, Ciro, and Mejía (2005), in which the cheese loses elasticity with ripening; this loss is probably due to protein degradation (Castañeda, 2002). Rha (1979) states that if a food is deformed under force, the

ACCEPTED MANUSCRIPT recovery is always less than the initial, and the degree of recovery will depend on the time interval in which the material was deformed, the rate at which stress was applied, the food mass, the moisture content and the composition; therefore, it is clear that the compositional changes previously discussed (Table 2) have an effect on the recoveries (Fig. 2) obtained in

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the cheeses studied.

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Table 4 shows the rheological parameters of the Burger´s model for both cheeses. The

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viscosities increased significantly (p<0.05) with increasing ripening times. The retarded compliances values (J1) remained constant for CFM and CMM during ripening,. Both

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cheeses presented higher instantaneous compliances (almost 33 and 16 times from CFM

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and CMM, respectively) than retarded compliances (J1
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proportional to the viscosity values because the compliance is the reciprocal of the applied

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stress (Jiménez-Avalos, Ramos-Ramírez, & Salazar-Montoya, 2005). Regarding the retarded compliance time (λret), it increases slightly from 2.34x10-2 to

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2.38x10-2 s for CFM and from 2.32x10-2 to 2.35x10-2 s for CMM; this shows the loss of

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elasticity of the cheeses as they ripen, as the retarded compliance time indicates the ability

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of cheeses to recover from the deformation (Osorio, Ciro, & Mejía, 2005).

4. Conclusions

This study provides a vision into the composition and dynamic rheological properties of Manchego-type cheeses made with the free and microencapsulated microorganisms undergoing ripening. The results show an influence of microencapsulation on the composition of cheeses, and such changes may lead to rheological changes. Although, both

ACCEPTED MANUSCRIPT cheeses showed viscoelastic rheological behavior, where the elastic component was higher than the viscous component (Gʼ>G”), statistical differences were observed (p<0.05) between the moduli of the cheeses during ripening, reflecting the effects of the microcapsules and ripening time on the storage and loss moduli. Among the rheological

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parameters evaluated in the Burger´s model, the viscosity, instantaneous compliance, and

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recovery percentages were dependent of the level of Manchego-type cheese ripening. The

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retarded time, the J0 and Ji values in the studied cheeses shows the loss of elasticity of the cheeses as they ripen. The differences in the composition during the ripening are mainly

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due to the effect of the microcapsules on the microorganisms used and the enzymes that

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they produce, which can directly affect the texture of the cheese. Due to the fact that the composition of the cheese was different after ripening, it is necessary, in subsequent

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studies, to evaluate the acceptability of the Manchego-type cheese with microencapsulated

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microorganisms through sensory evaluation studies. Acknowledgements

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The authors acknowledge the grant from CONACyT México to RGC and the technical

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References

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support from Eng. Miguel Márquez and Eng. Dolores Díaz.

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Özer, B., Kirmaci, H. A., Senel, E., Atamer, M., & Hayaloǧlu, A. (2009). Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in White-brined cheese by microencapsulation. International Dairy Journal, 19, 22-29. Piska, I., & Ŝtêtina, J. (2004). Influence of cheese ripening and rate of cooling of the processed cheese mixture on rheological properties of processed cheese. Journal of Food Engineering, 61, 551-555. Rosenberg, M., Wang, Z., Chuang, S. L., & Shoemaker, C. F. (1995). Viscoelastic properties changes in Cheddar cheese during ripening. Journal of Food Science, 60, 640-644. Rha, C. K. (1979). Viscoelastic properties of food as related to micro and molecular structures, Food Technology, 33, 71-75. Sanderson, G. R., & Clark, R. C. (1983). Gellan gum. Food Technology, 37, 63-70. Shoemaker, C. F., Nantz, J., Bonnans, S., & Noble, A. (1992). Rheological characterization of dairy products. Food Technology, 46, 98-104. Silva, H. L. A., Balthazar, C. F., Esmerino, E. A., Vieira, A. H., Cappato, L. P., Neto, R. P. C., …Cruz, A. G. (2017). Effect of sodium reduction and flavor addition on probiotic Prato cheese processing. Food Research International, Article in press. http://dx.doi.org/10.1016/j.foodres.2017.05.018 Tyle, P. (1993). Effect of size, shape and hardness of particles in suspension on oral texture and palatability. Acta Psychology, 83, 111-118. Van Vliet, T., Walstra, P. (1994). Water in casein gels; how to get it out or keep it in. Journal of Food Engineering, 22, 75-88. Venugopal, V., & Muthukumarappan, K. (2003). Rheological properties of Cheddar cheese during heating and cooling. International Journal of Food Properties, 6, 99-114. Visser, J. (1991). Factors affecting the rheological and fracture properties of hard and semihard cheese. Bulletin of the International Dairy Federation, 268. 49-61. Xiong, Y. L., & Kinsella, J. E. (1991). Influence of fat globule membrane composition and fat type on the rheological properties of milk based composite gels: II. Results Milchwisst, 46, 207-212. Yáñez, J., Salazar, J. A., Chaires, L., Jiménez, J., Márquez, M., & Ramos, E. G. (2002). Aplicaciones biotecnológicas de la microencapsulación. Avance y Perspectiva, 21, 313-319. Yáñez, F. J. (2007). Microencapsulación de Lactobacillus spp., empleando mezclas de biopolímeros de gomas arábiga, gelana y de semillas de mezquite, por polimerización interfacial. PhD Thesis, México City: Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional 1-247. Available from: library CINVESTAV- Zacatenco. Yvon, M., & Rijnen, L. (2001). Cheese flavour formation by aminoacid catabolism. International Dairy Journal, 11, 185-201.

ACCEPTED MANUSCRIPT Table and Figure captions Table 1 Population of microorganisms in Manchego-type cheeses during ripening. CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. Columns and rows with different letters are significantly different(p<0.05); mean ± standard deviation of 3 repetitions.

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Table 2 Composition of Manchego-type cheeses during ripening.

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RT = Ripening time (days). CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. (-) = dimensionless. Rows with different letters are significantly different (p<0.05); mean ± standard deviation of 3 repetitions.

Table 3 Behavior of the storage modulus, loss modulus and phase angle in Manchego-type cheeses.

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CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. (-) = dimensionless. Columns with different letters are significantly different (p<0.05).

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Table 4 Rheological parameters of the Burger´s model of Manchego-type cheeses.

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CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. J0 = Instantaneous compliance, Ji = retarded compliance, λret = retarded time. Columns with different letters are significantly different (p<0.05).

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Fig. 1. Behavior of dynamic moduli (Gʼ filled symbols, G” opened symbols) as a function of frequency in Manchego-type cheeses. a) Cheese with free microorganisms and b) cheese with microencapsulated microorganisms. 1 day (,), 15 days (,), 30 days (,), 45 days (,), and 60 days (,).

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Fig. 2. Creep and recovery curves in Manchego-type cheeses as a function of the ripening time. a) Cheese with free microorganisms and b) cheese with microencapsulated microorganisms.

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Fig. 1. Behavior of dynamic moduli (Gʼ filled symbols, G” opened symbols) as a function of frequency in Manchego-type cheeses. a) cheese with free microorganisms and b) cheese with microencapsulated microorganisms. 1 day (,), 15 days (,), 30 days (,), 45 days (,), and 60 days (,).

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Fig. 2. Creep and recovery curves in Manchego-type cheeses as a function of the ripening time. a) Cheese with free microorganisms and b) Cheese with microencapsulated microorganisms.

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Table 1 Population of microorganisms in Manchego-type cheeses during ripening. CFM

(days)

(log10CFUg-1)

1

7.80 ± 0.02ª

8.32 ± 0.00b

15

8.22 ± 0.06b

9.51 ± 0.04d

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Ripening time

45

PT

60

(log10CFUg-1)

8.57 ± 0.07c

9.67 ± 0.04e

8.81 ± 0.14d

9.82 ± 0.02f

8.51 ± 0.01c

9.96 ± 0.02g

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30

CMM

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Table 2 Composition of Manchego-type cheeses during ripening. RT Microorganisms Status

Fat

Protein

Moisture

Total solids

Ash

pH

Chlorides

(%)

(%)

(%)

(%)

(%)

T P

(-)

(%)

2.53±0.25ae

6.17±0.06a

2.50±0.02a

3.29±0.96b

5.82±0.04b

2.56±0.03a

I R

1

CFM

30.00±1.00a

24.55±0.77abf

45.28±0.13a

52.91±0.18a

15

CFM

30.33±0.57a

24.76±0.62a

44.69±0.52b

53.86±0.20b

30

CFM

29.33±0.57ab

24.51±0.62abf

44.52±0.34b

53.82±0.60b

2.84±0.26a

5.62±0.04c

2.61±0.04bc

45

CFM

28.33±0.57

cd

bcf

c

54.16±0.43bc

2.79±0.83ac

5.44±0.00d

2.68±0.02df

60

CFM

28.33±0.57cd

22.74±0.26cd

43.79±0.17c

55.25±0.49df

2.71±0.04ad

5.32±0.01e

2.75±0.03e

1

CMM

30.33±0.57a

25.96±0.51e

45.59±0.40d

54.54±0.45bd

2.50±0.02cde

6.50±0.05f

2.50±0.02a

15

CMM

30.66±0.57a

26.14±0.71e

44.85±0.16ab

54.54±0.51bd

3.68±0.12f

5.88±0.00b

2.56±0.02ab

30

CMM

28.66±0.57bc

24.70±0.29ab

44.88±0.12ab

54.94±0.70cd

3.43±0.07bf

5.50±0.00d

2.63±0.03cd

45

CMM

27.33±0.57de

23.81±0.77df

44.54±0.33b

55.25±0.76de

3.44±0.28bf

5.20±0.04g

2.74±0.03ef

60

CMM

26.33±0.57e

22.52±0.67g

44.51±0.62b

55.84±0.34ef

3.41±0.30bf

5.02±0.06h

2.79±0.05e

23.85±0.25

D E

T P

E C

C A

A

U N

43.78±0.73

M

C S

RT = Ripening time (days). CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. (-) = dimensionless. Rows with different letters are significantly different (p<0.05); mean ± standard deviation of 3 repetitions.

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tan δ

(Pa)

(-)

27000a

7130a

0.264

54700b

17900b

0.327

90600c

29400c

0.324

119000d

38800d

0.326

147000e

50300e

0.342

0.0846

85522f

25600f

0.299

0.0249

131455g

40660g

0.309

30

0.0497

221250h

71790h

0.324

45

0.2020

240000i

84200i

0.351

60

0.0611

348000j

119000j

0.341

1

0.0190

15

0.0346

30

0.0480

45

0.0474

60

0.0510

1

PT

15

M

CFM

AC

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CMM

CR

(Pa)

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Torque amplitude (mNm)

IP

G’’

US

G’

Ripening time (days)

ED

Chesse

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Table 3 Behavior of the storage modulus, loss modulus and phase angle in Manchego-type cheeses.

CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. (-) = dimensionless. Columns with different letters are significantly different (p<0.05).

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J0 (Pa-1)

J1 (Pa-1)

λret (s)

Recovery (%)

1

0.0113 0.0840

30

0.0412

45

0.0298

60

0.1270

5.56 X 10-5 a 3.52 X 10-5 b 2.54 X 10-5 c 2.58 X 10-5 d 1.05 X 10-5 e

1.00 X 10-6 a 1.00 X 10-6 a 1.00 X 10-6 a 1.00 X 10-6 a 1.00 X 10-6 a

2.34 X 10-2 a 2.35 X 10-2 a 2.35 X 10-2 a 2.37 X 10-2 a 2.38 X 10-2 a

70.67

15

2.83 X 106 a 4.36 X 106 b 8.81 X 106 c 1.30 X 107 d 2.65 X 107 e

1

0.0921

IP

T

Torque (mNm)

CR

CFM

Ripening time (days)

US

Cheese

66.98 60.45 58.33 48.63

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3.49 X 2.16 X 1.00 X 2.32 X 70.97 6f -5 f -6 a -2 a 10 10 10 10 15 0.0427 4.39 X 4.90 X 1.00 X 2.34 X 67.65 6g -5 g -6 a -2 a 10 10 10 10 CMM 30 0.0217 5.77 X 4.38 X 1.00 X 2.34 X 58.74 6h -5 h -6 a -2 a 10 10 10 10 45 0.0785 6.97 X 1.18 X 1.00 X 2.35 X 56.78 6i -5 i -6 a -2 a 10 10 10 10 60 0.0790 1.55 X 1.10 X 1.00 X 2.35 X 45.24 7j -5 j -6 a -2 a 10 10 10 10 CFM = Cheese with free microorganisms. CMM = Cheese with microencapsulated microorganisms. J0 = Instantaneous compliance, Ji = retarded compliance, λret = retarded time. Columns with different letters are significantly different (p<0.05).

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Graphical abstract

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Highlights  In this study, the rheological behaviors of Manchego-type cheeses were determined  The cheeses were prepared with microorganisms in the free status and microencapsulated  Gellan gum was used to microencapsulate Lactococcus lactis subsp lactis and cremoris  Both types of cheeses resulted in systems with predominantly elastic characteristics  The moduli Gʼ and G” increased with the microencapsulation and ripening time

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