«Foods 2013, 2, 100-119; doi:10.3390/foods2010100 OPEN ACCESS foods ISSN 2304-8158 Article Encapsulation of a Lactic Acid ...»
Foods 2013, 2, 100-119; doi:10.3390/foods2010100
Encapsulation of a Lactic Acid Bacteria Cell-Free Extract in
Liposomes and Use in Cheddar Cheese Ripening
Alice Beebyaanda Nongonierma 1, Magdalena Abrlova 1,2 and Kieran Noel Kilcawley 1,*
Teagasc, Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland;
E-Mails: firstname.lastname@example.org (A.B.N.); email@example.com (M.A.)
Department of Dairy and Fat Technology, Institute of Chemical Technology, Prague Technika5, Prague 6, 16628, Czech Republic * Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org;
Tel.: +353-025-42245; Fax: +353-025-42340.
Received: 20 January 2013; in revised form: 5 March 2013 / Accepted: 6 March 2013 / Published: 13 March 2013 Abstract: A concentrated form of cell free extract (CFE) derived from attenuated Lactococcus lactis supsb. lactis 303 CFE was encapsulated in liposomes prepared from two different proliposome preparations (Prolipo Duo and Prolipo S) using microfluidization. Entrapment efficiencies of 19.7 % (Prolipo S) and 14.0 % (Prolipo Duo) were achieved and the preparations mixed in the ratio 4 (Prolipo Duo):1 (Prolipo S).
Cheddar cheese trials were undertaken evaluating the performance of CFE entrapped in liposomes, empty liposomes and free CFE in comparison to a control cheese without any CFE or liposomes. Identical volumes of liposome and amounts of CFE were used in triplicate trials. The inclusion of liposomes did not adversely impact on cheese composition water activity, or microbiology. Entrapment of CFE in liposomes reduced loss of CFE to the whey. No significant differences were evident in proteolysis or expressed PepX activity during ripening in comparison to the cheeses containing free CFE, empty liposomes or the control, as the liposomes did not degrade during ripening. This result highlights the potential of liposomes to minimize losses of encapsulated enzymes into the whey during cheese production but also highlights the need to optimize the hydrophobicity, zeta potential, size and composition of the liposomes to maximize their use as vectors for enzyme addition in cheese to augment ripening.
Foods 2013, 2 101 Keywords: Cheddar cheese; liposomes; cell-free extract; encapsulation; sensory
1. Introduction Acceleration of cheese ripening has been proposed as a way to produce a fast ripening curd for processed cheese or to reduce costs associated with cheese manufacture . Different strategies for acceleration of cheese ripening have been described with the addition of exogenous enzymes being the most studied technique [1–3]. The addition of exogenous enzymes is an accepted method to accelerate Cheddar cheese ripening, however significant amounts of enzyme are lost to the whey and can have adverse effects on whey quality . Hence, the use of encapsulated enzymes has evolved as a method to combat losses to whey  and improve enzyme retention in the curd . Previous studies have identified liposomes as suitable vectors for the inclusion of enzymes into cheese as they have a high affinity for milk fat and can encapsulate sufficiently large quantities of water soluble material .
Previous studies have described the acceleration of cheese ripening with enzymes encapsulated in liposomes [7–13]. Food-grade water soluble enzymes can be encapsulated in liposomes using milk fats or different food-grade proliposomes [6,14], and losses to whey may be minimized as liposomes partition with the fat globules and the casein matrix when added to the milk . Microfluidization, is a homogenization method based on the use of relatively high pressures, and has been described in the manufacture of liposomes [7,14–16]. In contrast with other liposome preparation methods, microfluidization does not require organic solvents and is easy to scale up, making it suitable for large scale food grade applications [7,15,17].
The objectives of this study were to evaluate the impact of the addition of cell-free extracts (CFE) of Lactococcus lactis subsp. lactis 303 and liposome-encapsulated CFE of Lactococcus lactis subsp.
lactis 303 on the ripening of Cheddar cheese. CFE of Lactococcus lactis subsp. lactis 303 was produced by microfluidization and subsequently encapsulated in two liposome preparations with different phospholipid compositions. Losses of key intracellular enzymes were monitored during production and ripening together with, microbiological analyses and physico-chemical characteristics of the cheeses (composition, water activity and microscopy). Descriptive sensory analysis and volatile profiling were also determined at 112 days ripening.
2. Results and Discussion
2.1. Lactococcus lactis subsp. lactis 303 CFE Manufacture and Encapsulation in Liposomes
In the RSM after growth, low level of PepX activity was determined but no LDH activity was detected indicating minimal lysis of the bacterial cells at this stage (Table 1). After one pass through the microfluidizer, there was a significant increase in both PepX and LDH activities (p 0.05) and ~18% reduction of viable cells due to cell disruption of Lactococcus lactis subsp. lactis 303 cells. The total cell counts, PepX and LDH activity increased in the microfluidized cells after freeze drying due to a concentration effect and additional cell lysis as cell counts and enzyme activities are expressed on weight basis rather than on a volume basis. No viable cells were present in the freeze dried CFE as anticipated and the level of Pep X activity was very high due to the fact that it was concentrated in the cell extract (Table 1). This result also highlights the fact that it is possible to freeze dry CFE and maintain peptidase activity.
Encapsulation of Lactococcus lactis subsp. lactis 303 CFE was undertaken in two types of proliposomes (Duo and S). The amount of PepX activity in the water soluble 303 CFE used to manufacture the Cheddar cheese samples is given in Table 2. The Prolipo Duo preparation has a lower zeta potential than Prolipo S (Table 2) preparation because it contains more negatively charged phospholipids. Lower zeta potential values are associated with greater liposomal stability. A liposome with a low zeta potential can cause electrostatic repulsions, which in turn may prevent destabilization processes, such as coalescence and aggregation . During the encapsulation process, a substantial amount of CFE remained unbound to the liposomes (70%). The encapsulation efficiency of 303 CFE was 19.7% for Prolipo S and 14.0% for Prolipo Duo (Table 2) which was not significantly different (p ≥ 0.05). Encapsulation efficiency of CFE in liposomes made with Prolipo S up to 58.4% has been reported for a cell free extract of Lactobacillus casei subsp. pseudoplantarum  and lower encapsulation efficiency in liposomes of 12.7% for cryopsin has been described .
Nongonierma et al.  reported enzyme entrapment efficiencies of 62.7% in Prolipo C and 29.2% in Prolipo S for Debitrase DBP20. In Prolipo VPF 012, encapsulation efficiencies of 32%–36% have been reported for bacterial and fungal proteinases , 35.9% for Palatase M and 40.3% for Lipase 50 . Liposomal encapsulation as low as 12.7% for cryopsin was shown to accelerate proteolysis in Manchego cheese , therefore the encapsulation efficiencies achieved in this study Foods 2013, 2 103 were deemed appropriate to positively influence acceleration of proteolysis and thus flavor in Cheddar cheese.
The final preparation used in the cheese-making trials contained a mixture of both Proliposome preparations at a ratio of 4 parts Prolipo Duo and 1 part Prolipo S. This mixture was utilized as the Prolipo Duo liposomes are more hydrophobic than the Prolipo S and should partition better with the milk fat globule membrane and the casein matrix during cheese production increasing retention in the cheese curd [1,6]. It has been suggested that liposomes are distributed in the curd in the same fashion as bacterial cells , where liposomes behave as a carrier for different enzyme activities similarly to bacterial cells. However, it is believed that enzyme release from liposomes occurs at a faster rate than from bacterial cells [3,18]. Although the mechanisms of enzyme release from liposomes in cheese are poorly understood . It has been suggested that enzyme release from liposomes in cheese may involve various parameters including temperature, pH and ionic strength . In addition, it is thought that liposome degradation in cheese may occur following aggregation processes which are favored at low pH values. It has been shown that a decrease from pH 7 to 5 was responsible for the release of active agent (calcein) encapsulated in liposomes . Several studies have demonstrated the potential of liposome encapsulated enzymes as a means to accelerate cheese ripening [10–12], by reducing losses to whey .
2.2. Influence of CFE and Encapsulated CFE on the Composition and Water Activity of the Cheeses
Some textural defects have also been associated with addition of liposomes into cheeses, which is thought to be due to an increase in cheese moisture [7,8,10–12]. An increase in the cheese moisture has been associated with water binding at the liposome surface . In addition to the increased moisture, the associated decrease in the protein content can lead to a less firm and more brittle cheese structure . In contrast, Lariviere et al.  showed that apart from increased moisture levels, cheeses with liposomes did not have any negative textural issues in comparison to a control cheese without liposome addition. No significant differences (p ≥ 0.05) were noted in water activity between the cheeses, highlighting that not only was there no differences in moisture, but that water activity was not altered by the inclusion of liposomes. Lower water activity is known to reduce rates of proteolysis in cheese .
2.3. Enumeration of Starter and Non-Starter Lactic Acid Bacteria and Enzyme Activities during Cheese Ripening Evolution of LAB and NSLAB were monitored during the ripening in each cheese (Figure 1). As anticipated, LAB counts (Figure 1a) decreased and NSLAB increased during ripening (Figure 1b). The decrease in LAB has been attributed to the changes in the cheese matrix including reduction in pH, lactose content and an increase in salt concentration . There were no significant differences in the cell counts determined for the four cheeses both for LAB and NSLAB (p ≥ 0.05).
Foods 2013, 2 105 Figure 1. Microbiological count in different cheeses for (a) lactic acid bacteria (LAB) and (b) non-starter lactic acid bacteria (NSLAB) as a function of ripening time. Each point is the average of three determinations (n = 3). Cheese 1, Control; Cheese 2, cheese with empty liposomes S and Duo; Cheese 3, cheese with liposomes S and Duo containing the encapsulated Lactococcus lactis ssp. lactis 303 cell-free extract (CFE); Cheese 4, cheese with Lactococcus lactis ssp. lactis 303 CFE.
PepX and LDH activities were measured in the curd and whey samples during production of each cheese. Only residual activities for LDH from starter LAB were found as anticipated (data not reported), as cell lysis does not normally occur in the early stages of cheese making . There were no significant differences between PepX activities of the different curd samples (p ≥ 0.05), but there were significant differences (p 0.05) in PepX activity in the whey samples (Figure 2).
Foods 2013, 2 106 Figure 2. Post-proline dipeptidyl aminopeptidase (PepX) in the curd and whey from the different cheese samples. Each point is the average of three PepX determinations (n = 3).
For the whey or curd samples, figures with different letters are significantly different (p 0.05). Cheese 1, Control; Cheese 2, cheese with empty liposomes S and Duo;
Cheese 3, cheese with liposomes S and Duo containing the encapsulated Lactococcus lactis ssp. lactis 303 cell-free extract (CFE); Cheese 4, cheese with Lactococcus lactis ssp. lactis 303 CFE.
Significantly higher PepX activities were measured in the whey in Cheeses 3 and 4, compared to Cheeses 1 and 2 (p ≥ 0.05). As Cheeses 1 and 2 did not contain additional CFE it was anticipated that levels should be similar and lower than Cheeses 3 and 4. The fact that levels of Pep X are significantly higher (p 0.05) in Cheese 4 highlights that significant amounts of the added free CFE were lost to the whey at drainage. The fact that additional levels of PepX activity were not found in curds and that lower levels were in the whey in Cheese 3 in comparison to Cheese 4 indicates that additional CFE is incorporated into the curd within the liposome preparations.
PepX and LDH activities were monitored in the different cheeses over 112 days of ripening. There were no significant (p ≥ 0.05) differences between the four cheese for PepX or LDH activity. Both LDH and PepX (Figure 3a,b) activities increased numerically during ripening. LDH levels increased up to day 56 and then dropped up to day 84 and increased again up to day 112 in all cheeses (Figure 3a). The initial increase in LDH activity is likely related to lysis of starter LAB (Figure 1a), and the later increase presumably due to a combination of continued lysis of starter LAB and lysis of NSLAB. Even though NSLAB were seen to numerically accumulate (Figure 1b) at this time point, it is anticipated that a percentage will also autolyse . PepX activity increased rapidly between day 0 and 28 and then leveled off (Figure 3b).
Foods 2013, 2 107 Figure 3. (a) Lactate dehydrogenase (LDH) activity in the different cheese samples as a function of ripening time. (b) Post-proline dipeptidyl aminopeptidase (PepX) activity in the different cheese samples as a function of ripening time. Each point is the average of three determinations (n = 3). Cheese 1, Control; Cheese 2, cheese with empty liposomes S and Duo; Cheese 3, cheese with liposomes S and Duo containing the encapsulated Lactococcus lactis ssp. lactis 303 cell-free extract (CFE); Cheese 4, cheese with Lactococcus lactis ssp. lactis 303 CFE.