Meat Science 111 (2016) 198–203

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Meat Science

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Use of alginate beads as carriers for lactic acid bacteria in a structured

system and preliminary validation in a meat product
Maria Rosaria Corbo, Antonio Bevilacqua ⁎, Barbara Speranza, Barbara Di Maggio,
Mariangela Gallo, Milena Sinigaglia
Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: This paper proposes the microencapsulation into alginate beads of 4 isolates of lactic acid bacteria
Received 2 October 2014 (Lactobacillus spp.), previously isolated from pork meat. First, the beads were studied in relation to the en-
Received in revised form 18 August 2015 capsulation yield (EY), kinetic of cell release in a structured system, and survival throughout bead storage at
Accepted 8 October 2015
4 °C. EY was 93–96% and the survival of the encapsulated microorganisms was variable, with two isolates
Available online 13 October 2015
showing a bacterial population of 6.1–6.9 log cfu/g after 35 days under refrigerated conditions.
Keywords:
Thereafter, the paper addressed a preliminary validation in a meat model system, containing salt, nitrites
Lactic acid bacteria and nitrates, lactose, pepper, and then in a commercial preparation of pork meat. For the validation in
Alginate pork meat, free cells were used as controls.
Beads Cell released from beads were able to achieve a significant acidification; in particular, after 7 days they showed
Release the same results of free cells.
Acidification © 2015 Elsevier Ltd. All rights reserved.
Structured system

1. Introduction Sabikhi, Babu, Thompkinson, & Kapila, 2010), lactococci (Dianawati,

The interest towards microencapsulation increased significantly
for its promising application to design suitable carriers for probiotics
or as a way to improve food fermentation through the optimization
of re-usable bio-catalysts (Corbo, Bevilacqua, Gallo, Speranza, &
Sinigaglia, 2013; Gallo, Bevilacqua, Speranza, Sinigaglia, & Corbo,
2014).
Microencapsulation helps to separate a core material from its envi-
ronment until it is released, thereby improves its stability, extends the
core's shelf life and provides a sustained and controlled release, masking
flavors, colors or odors (Kailasapathy, 2002).
The most commonly reported microencapsulation procedure
is based on calcium-alginate gel capsule formation. Spherical
polymer beads with diameters ranging from 0.3 to 3.0 mm and
immobilizing active biomass are produced using extrusion with sy-
ringe and stirred calcium bath; alginate is used in concentration
range of 0.5–4% (Burgain, Gaiani, Linder, & Scher, 2011; Corbo
et al., 2013).
Different microorganisms were encapsulated in alginate: lactobacilli
(Nualkaekul, Lenton, Cook, Khutoryanskiy, & Charalampopoulos, 2012;
Mishra, & Shah, 2013), bifidobacteria (Fritzen-Freire et al., 2012), and
Saccharomyces cerevisiae (Gallo, Bevilacqua, Speranza, Sinigaglia &
Corbo, 2014). Many authors reported that entrapped cells could start
a fermentation in yoghurt (Picot & Lacroix, 2004); cheese (Özer,
Kirmaci, Şenel, Atamer, & Hayaloğlu, 2009), ice cream (Prevost &
Divies, 1992), mayonnaise (Khali & Mansour, 1998), soy milk (Corbo
et al., 2013), and dry fermented sausages (Muthukumarasamy &
Holley, 2006).
The use of beads was proposed in the past for liquid media; to the
best of our knowledge, few data are available on the use of capsules in
a structured system. This paper addressed the microencapsulation into
alginate beads of 4 isolates of Lactobacillus spp., isolated from pork
meat and selected for their technological (acidification, growth in
wide ranges of temperature, salt concentration, pH values) and probiot-
ic traits (survival in the presence of bile salt and at pH 2.0, bioactivity to-
wards foodborne pathogens, hydrophobicity and auto-aggregation,
antibiotic resistance/sensitivity). The isolates were chosen as repre-
sentative of promising functional starter cultures to design a biocat-
alyst (alginate beads with microorganisms) intended for sausage
fermentation.
The main topic (use of beads in structured systems) was addressed
through some intermediate milestones:

a) Assessing encapsulation yield and viability of encapsulated cells

⁎ Corresponding author. throughout storage.
E-mail address: antonio.bevilacqua@unifg.it (A. Bevilacqua). b) Studying cell release from the beads into a structured and solid

http://dx.doi.org/10.1016/j.meatsci.2015.10.005
0309-1740/© 2015 Elsevier Ltd. All rights reserved.
 M.R. Corbo et al. / Meat Science 111 (2016) 198–203
system.
c) Evaluating bead performances (acidification) in a model system
and then in a pork-meat preparation, as a function temperature
and sausage ingredients (nitrites, nitrates, lactose, pepper).
2. Materials and methods
2.1. Microorganisms
Four isolates of Lactic Acid Bacteria (LAB) isolated from pork meat
and characterized for their technological and probiotic traits were
used throughout this research. The isolates (162, 178, 204, 239) were
identified as Lactobacillus plantarum (Bevilacqua, unpublished results)
and stored at − 20 °C in MRS broth (Oxoid, Milan, Italy) added with
33% of sterile glycerol (J.T. Baker, Milan, Italy). Before each assay the mi-
croorganisms were grown in MRS broth incubated at 37 °C for 24 h.
2.2. Microencapsulation
Alginate beads were produced as reported by Corbo et al. (2013).
Bacterial cultures were centrifuged at 1000 g for 10 min; then, the
cells were washed with sterile distilled water (cell suspension).
20 ml of cell suspension (8–9 log cfu/ml) was added with Na-alginate
2% w/v (Carlo Erba, Milan, Italy) and mixed gently for 1–3 min. After
2–3 h the gel was manually extruded through a sterile 10-ml-syringe
and the capsules dipped for ca. 5 min in a CaCl2 solution (0.50% and
8.00% w/v) (J.T. Baker).
The encapsulation yield (EY) was evaluated as follows: 5 g of beads
was diluted with 50 ml of a sterile 0.1 M sodium citrate solution (J. T.
Baker) and homogenized through a laboratory blender, to achieve the
complete dissolution of capsules, as described by Chávarri et al.
(2010). Both bead homogenates and cell suspension (i.e., the original
cell suspension used to prepare beads) were serially diluted in a saline
solution (0.90% NaCl) and bacterial population was evaluated through
pour plating on MRS agar (incubated at 30 °C for 48 h under anaerobic
conditions in a jar by using the kit Anaerogen from Oxoid).
EY was evaluated as follows:
 
Nbead
EY ¼  100
Nsuspension
where Nbead (expressed as cfu/g) and Nsuspension (expressed as cfu
per gram of solution) are the viable counts in the beads and in cell
suspension, respectively.
2.3. Bacterial population within bead storage
Beads were put into an empty sterile plate and stored at 4 °C and an-
alyzed periodically (7, 14, 21, 35 days) for the evaluation of bacterial
population through the pour plate method on MRS Agar.
2.4. Kinetic of cell release from capsules in agar medium
0.40 g of beads was put in sterile Petri plates and mixed with 20 ml of
a 1.20% sterile liquid medium (Agar technical n. 3, Oxoid, autoclaved
and cooled to 45–50 °C); beads were placed into the plates after medi-
um pouring. Then, the medium was gently mixed, let to solidify; after
solidification the plates were stored at 25 °C and analyzed periodically
(immediately after sample preparation and after 2, 5, 7, 12, 14,
21 days) to evaluate the amount of cells released from beads. The agar
was gently removed from the plates, diluted (1:10) with a sterile saline
solution (0.90% NaCl), and homogenized through a Stomacher for 30 s.
This kind of homogenization did not disrupt or affect the integrity of
alginate beads. The viable count was assessed through the pour plate
method.
199
Table 1
Meat broth.
Ingredient Amount (g/l)
Beef extract (Oxoid) 5.00
Bacteriological peptone (Oxoid) 10.00
Tryptone (Oxoid) 5.00
Agar (Oxoid) 12.00
Nitrites (C. Erba, Milan) Variable
Nitrates (C. Erba) Variable
NaCl (C. Erba) Variable
Lactose (C. Erba) Variable
Black pepper (C. Erba) Variable
2.5. Meat model system: validation of microencapsulation
Beads were loaded into a meat broth (Cardenas, Giannuzzi, &
Zaritzky, 2007), slightly modified as reported in Table 1. The medium
was sterilized at 121 °C for 15 min; then, at 50 °C 0.40 g beads per
20 ml of medium was added.
The amounts of nitrites, nitrates, lactose, sugar, and black pepper,
and the storage temperature varied according to a 3k − p Fractional
Factorial Design (Table 2). The factors of the design were combined as
reported by Box, Hunter, and Hunter (2005).
The samples were stored at 20 and 30 °C. Bacterial population and
pH were evaluated after 24 and 48 h through the pour plate method
and a Crison pH-meter (Crison Instruments, Barcelona, Spain),
respectively.
2.6. Lab-scale validation
This assay was carried out using the isolate 178. Alginate beads were
produced as previously described and added to a commercial homoge-
nized infant preparation of pork meat (1.40 g of capsules for 70 g of
product). The composition was as follows: pork meat, 40.00%; salt,
0.50%; rice flour, 20.00%; and water. Aliquots of commercial preparation
inoculated with free cells (i.e., not encapsulated cells, 8 log cfu/g) were
used as control samples. The samples were added with nitrites, nitrates,
lactose, and black pepper, and stored at 20 or 30 °C, according to the
Fractional Design reported in Table 2. Bacterial population and pH
were evaluated after 2, 5, and 7 days of storage.
2.7. Statistical analysis and data modeling
The experiments were performed over two different batches; for
each batch duplicate measurements were done. The results were ana-
lyzed through one-way ANOVA and Tukey's test as the post-hoc com-
parison test (P b 0.05); paired comparisons (i.e. difference between 2
samples) were analyzed through t-test. Moreover, data from the exper-
iments on performances of encapsulated cells were modeled using the
theory of DoE (Design of Experiments); bacterial population, acidifica-
tion (decrease of pH), and pH were set as dependent variables whereas
nitrites, nitrates, lactose, temperature, and pepper were used as inde-
pendent variables. The statistical analyses were performed through
the software Statistica for Windows (StatSoft, Tulsa, OK.).
3. Results
3.1. Performances of microencapsulation
Encapsulation yield of beads (EY) was 92.90–96.80% (data not
shown), without any significant difference amongst the isolates (P b
0.05). Concerning the survival of the isolates, the initial bacterial popu-
lation was 9.00 ± 0.30 log cfu/g; however, the trend within the storage
relied upon the microbial target, as the isolate 204 experienced a fast
death kinetic and decreased until the detection limit after 14 days. On
200 M.R. Corbo et al. / Meat Science 111 (2016) 198–203

Table 2
Factorial Fractional Design (3k − p) for the amounts of nitrites, nitrates, salt, lactose and black pepper, and storage temperature for the preliminary validation in the model system and for
bead validation in the commercial pork product. Coded levels: −1, minimum value; 0, average value; +1, maximum value.

Coded values Real values

Na-nitrites Na-nitrates NaCl T Lactose Black pepper Na-nitrites Na-nitrates NaCl (%) T Lactose (%) Black pepper (%)
(ppm) (ppm) (°C)

A −1 −1 −1 1 −1 1 0.00 0.00 0.00 30 0.00 0.10

B 1 −1 −1 1 1 1 150.00 0.00 0.00 30 0.40 0.10
C −1 1 −1 1 1 −1 0.00 250.00 0.00 30 0.40 0.00
D 1 1 −1 1 −1 −1 150.00 250.00 0.00 30 0.00 0.00
E −1 −1 1 1 1 −1 0.00 0.00 5.00 30 0.40 0.00
F 1 −1 1 1 −1 −1 150.00 0.00 5.00 30 0.00 0.00
G −1 1 1 1 −1 1 0.00 250.00 5.00 30 0.0 0.10
H 1 1 1 1 1 1 150.00 250.00 5.00 30 0.40 0.10
I 0 0 0 0 0 0 75.00 150.00 2.50 20 0.20 0.05

the other hand, the isolates 162, 178 and 239 showed a tailing effect
with a residual count after 35 days of 6.10–6.90 log cfu/g for isolates
178 and 162 and ca. 4.5 log cfu/ml for the isolate 239 (Fig. 1).
As a final step we evaluated the kinetic of bacterial release in a model
solid system. The beads released cells up to 6.00 log cfu/g after 2 days;
thereafter the amount of released cells did not undergo to a further in-
crease (P N 0.05) (data not shown).
3.2. Performance of beads into a synthetic system
A preliminary validation of beads was performed in a structured
model system, containing agar and some ingredients to simulate meat.
Fig. 2 shows the actual values of acidification (difference between initial
and final pHs) experienced in the different combinations of the design
after 48 h. The combinations B (nitrites, 150 mg/kg; temperature, 30 °C;
lactose, 0.40%; black pepper, 0.10%) and C (nitrates, 250 mg/kg; temper-
ature, 30 °C; lactose, 0.40%) showed the highest acidification, correspond-
ing to reduction of pH of ca. 2.60. Significant levels of acidification (ΔpH,
1.0–1.7) were also recovered in the samples E (NaCl, 5%; temperature, 30
°C; lactose, 0.40%), H (nitrites, 150 mg/kg; nitrates, 250 mg/kg; NaCl, 5%;
temperature, 30 °C; lactose, 0.40%; black pepper, 0.10%) and I (nitrites,
75 mg/kg; nitrates, 125 mg/kg; NaCl, 5%; temperature, 20 °C; lactose,
0.20%; black pepper, 0.05%). These values were used as input data to
Fig. 1. Viability of lactic acid bacteria (isolates 162, 178, 204, 239) throughout bead storage
at 4 °C. Mean values ± standard deviation. The letters indicate significant differences (one-
way ANOVA and Tukey's, P b 0.05).
run a black-box model, using the theory of DoE; acidification was the out-
put of this model (dependent variable), while the factors of the design
were used as independent variables. The main result of DoE theory is a
set of standardized effects, which show how a factor affects the output
(in a positive or in a negative way) and if it is significant or not. When
running a black-box approach as in DoE theory, the main output is a poly-
nomial equation, where for each individual, quadratic or interactive ef-
fects of the factors of the design there is a mathematical coefficient and
a standard error. The ratio mathematical coefficient vs standard error is
a standardized effect. If this value is N than a critical t-test (generally
2.10–2.40), the effect is significant. The standardized effects give two
main details: if a factor is significant and which is the most significant/im-
portant factor (Box et al., 2005).
The standardized effects of lactose, salt, pepper and preservatives on
acidification in the model system are reported in Table 3; as expected,
the only factor playing a role was lactose, as it was used as a carbon
source for fermentation. Viable count was not affected by the factors
of the design, as we found a bacterial population of 7.40–7.80 log cfu/g
in all the samples (P N 0.05) (data not shown).
3.3. Lab-scale validation
Finally, beads were tested as carriers of the isolate 178 for the fer-
mentation of a pork infant formula; although the isolates experienced
similar performances in vitro with high EYs, the isolate 178 was selected
as it was more vigorous and showed higher value of auto-aggregation
(i.e. competition against foodborne pathogens) in a preliminary re-
search (Bevilacqua, unpublished results). Fig. 3 shows the acidification
expressed by both free and encapsulated cells after 2 days. In the com-
binations A (temperature, 30 °C, black pepper, 0.10%), E (NaCl, 5%; tem-
perature, 30 °C; lactose, 0.40%), and H (nitrites, 150 mg/kg; nitrates,
250 mg/kg; NaCl, 5.00%; temperature, 30 °C; lactose, 0.40%; black pep-
per, 0.10%), ΔpH for free cells was higher than that reported for the en-
capsulated ones; otherwise, after 7 days the differences were not
significant. As reported above these values were used for a DoE ap-
proach to evaluate the standardized effect; the outputs are reported in
Table 4. Despite the preliminary validation in the model system, the
acidification of the isolate 178 was affected by lactose as positive term
and by nitrites and nitrates as negative terms after 5 and 7 days, i.e. acid-
ification increased by increasing the amount of lactose and decreased by
adding nitrites and nitrates; a mathematical positive effect was also
found for pepper, thus confirming its positive effect on metabolism, as
reported in the past by Bacus (1984).
Finally, Fig. 4 reports the average bacterial population both for free
and encapsulated cells; after 2 days, the samples containing the alginate
beads showed a lower viable count than the sample inoculated with free
cells (8.19 vs 9.13 log cfu/g), but the differences were not significant
throughout storage (P N 0.05).
 M.R. Corbo et al. / Meat Science 111 (2016) 198–203 201

Fig. 2. pH decrease after 48 h in the agar medium containing the beads with LAB (isolates 162, 178, 204, 239). Mean values ± standard deviation. The letters indicate significant differences
amongst the different combinations of the design for each isolate (one-way ANOVA and Tukey's, P b 0.05). The results of statistic are the same for all the isolates. A, pepper; B, nitrite +
pepper + lactose; C, nitrate + lactose; D, nitrite + nitrate; E, lactose + salt; F, nitrite + salt; G, nitrate + pepper + salt; H, nitrite + nitrate + lactose + pepper + salt. For these samples
the factors were set as follows: nitrite, 150 ppm; nitrate, 250 ppm; salt, 5%; lactose, 0.4%; pepper, 0.1%; temperature, 30 °C (level “+1” of the design). The sample I was the central point,
with the variable set at the level “0”: nitrite, 75 ppm; nitrate, 150 ppm; salt, 2.5%; lactose, 0.4%; pepper, 0.05%; temperature, 20 °C.

4. Discussion this approach, as they survived into the beads for at least 35 days with
a pronounced tailing effect and a residual population of 6.10–6.90 log cfu/g,
The evaluation of encapsulation efficiency is a complex process and in agreement with the data concerning the viability of the entrapped cells
relies upon many different parameters, like the ability of beads to entrap reported by many authors (Champagne & Fustier, 2007; Chávarri et al.,
cells, cell survival throughout bead storage and cell release after 2010; Corbo et al., 2013; Lee, Cha, & Park, 2004; Anal & Singh, 2007). The
immobilization. These parameters were evaluated respectively through last parameter to assess the suitability of the microencapsulation into alginate
encapsulation yield (EY), assessment of bacterial population into beads was the kinetic of cell release in a solid system; generally, the
alginate beads when stored at 4 °C and release of cells from beads in a evaluation of the kinetic of cell release was performed in liquid media
structured system. The values of EY (92.9–96.8%) were in line with (Bevilacqua et al., 2011; Corbo et al., 2013) and to the best of our knowledge
those reported by Corbo et al. (2013) (EY of ca. 93%) for the encapsula- this paper was the first attempt to define cell release in a structured medium.
tion of L. plantarum into 2%-alginate beads. The yield relies upon many The results suggested that cell release occurred within 48 h, thereafter
factors, but above all on the kind of polymer and its concentration: for the system attained a plateau corresponding to a critical viable count of ca.
example, Bevilacqua, Corbo, and Sinigaglia (2011) found EY of 54.8% 6–7 log cfu/g.
for Lactobacillus reuteri and 83.33% for Lactobacillus rhamnosus loaded The beads were generally proposed in the past as suitable carriers for
into 10% alginate beads, while Chávarri et al. (2010) reported for probiotics into the gut, although some authors reported on a possible
Bifidobacterium bifidum and Lactobacillus gasseri respectively an EY of use as biological catalyst to start a fermentation and protect the cells
40.20% and 39.20% in alginate-chitosan beads; finally, Picot and from the environment and/or as re-usable system (Corbo et al., 2013;
Lacroix (2004) found an efficiency of 25.57%, when bifidobacteria Özer et al., 2009; Picot & Lacroix, 2004; Prevost & Divies, 1992; Khali &
were encapsulated in whey protein. Mansour, 1998). In this paper we explore the possible use of beads as
The results of cell viability suggested a strong microorganism- an alternative way to add a starter culture to sausage. First, beads
dependence and the isolates 178 and 162 were the most suitable for were added to a controlled system (a structured lab medium) to evalu-
ate the ability of the microorganisms released by beads to start the fer-
mentation; thereafter, the beads containing the isolate 178 were added
to a meat product to explore bead ability to start the acidification of a
Table 3
Statistical effects of nitrites, nitrates, salt, temperature, lactose, and pepper on the acidifi-
real system.
cation expressed by lactic acid bacteria in meat broth after 48 h. –, not significant; R2ad, R2 The effects of the parameters of the design (lactose, nitrites, nitrates,
adjusted for the multiple regression. pepper, salt) were different in the model and in the real system: in the
lab medium lactose acted as a single variable leading the acidification,
Isolate 162 Isolate 178 Isolate 204 Isolate 239
while meat preparation was a complex system, thus the effects of the
Nitrites – – – –
other parameters (nitrites, nitrates, salt) become important for a possi-
Nitrates – – – –
NaCl – – – – ble strong interaction of these ingredients with the matrix.
Temperature – – – – The factors of the design (i.e. the ingredients of the mix) affected the
Lactose 5.22 4.55 4.12 4.51 acidification, but not cell release, thus suggesting that it could be influ-
Pepper – – – – enced by polymer and its concentration, kind of loaded microorganism,
R2ad 0.892 0.864 0.841 0.861
as well as the structure of the system (solid or liquid), as the kinetic
202 M.R. Corbo et al. / Meat Science 111 (2016) 198–203

Fig. 4. Box-whisker plot for the average viable count of lactic acid bacteria in the sample of
meat preparation inoculated with free cells (free) or added with alginate beads (bead)
after 2, 5, and 7 days (t2, t5, t7). SD, standard deviation. The letters indicate significant dif-
ferences (one-way ANOVA and Tukey's test, P b 0.05).

higher, above all in some combinations of the design (samples A, E,

Fig. 3. Decrease of pH in meat products added with beads or inoculated with free cells after
2 (a) and 7 (b) days. Mean values ± standard deviation. The symbol “*” indicates a signif-
icant difference between beads and free cells (t-test, P b 0.05). A, pepper; B, nitrite + pep-
per + lactose; C, nitrate + lactose; D, nitrite + nitrate; E, lactose + salt; F, nitrite + salt; G,
nitrate + pepper + salt; H, nitrite + nitrate + lactose + pepper + salt. For these samples
the factors were set as follows: nitrite, 150 ppm; nitrate, 250 ppm; salt, 5%; lactose, 0.4%;
pepper, 0.1%; temperature, 30 °C (level “+1” of the design). The sample I was the central
point, with the variable set at the level “0”: nitrite, 75 ppm; nitrate, 150 ppm; salt, 2.5%;
lactose, 0.4%; pepper, 0.05%; temperature, 20 °C.
reported in this paper was quite different from that suggested by Corbo
et al. (2013) for the same kind of beads incorporated in a liquid medium.
The last output was found throughout bead validation in the real
system; after 2 days, the average acidification of free cells was
Table 4
Statistical effects of nitrites, nitrates, salt, temperature, lactose, and pepper on the acidifi-
cation expressed by the isolate 178 loaded into alginate beads after 2, 5 and 7 days, valida-
tion in the commercial pork-infant formula. –, not significant; R2ad, R2 adjusted for the
multiple regression.
Sampling intervals
2 days 5 days 7 days
and H), due probably to a higher viable count; on the other hand,
after 7 days the microencapsulated cells achieved the same level of
acidification as the free ones. In addition, the average value of bacte-
rial population of free cells showed a negative trend, while the viable
count did not undergo any significant changes in the samples
containing the beads.
The use of microencapsulated starter was already proposed in the
past; for example, Muthukumarasamy and Holley (2006) used encapsu-
lated L. reuteri in meat preparations and found the same level of acidifi-
cation as in the control samples, containing the free cells; in addition,
they recovered a significant reduction in the viable count of the probiot-
ic in the controls, otherwise at the end of fermentation the decrease was
only 0.50 log cfu/g in the samples containing the beads.
5. Conclusion
This paper proposes the microencapsulation into alginate beads of
some isolates of lactic acid bacteria, selected from pork meat for their
technological and probiotic traits. The protocol of microencapsulation
was studied in relation to encapsulation yield (ca. 93–96%), viability of
cells into the beads (at least 35 days for 2 isolates) and cell release in a
structured system. Then, beads were used for a preliminary validation
in a lab medium and thereafter in a commercial preparation containing
pork meat; cells released from beads were able to achieve a significant
acidification of the samples and after 7 days they showed the same
results of free cells.
The results of this paper suggested that the microencapsulation
could be a promising way to design new kind of starter cultures for
meat. Alginate beads, in fact, could assure the release of the microbial
targets in a solid system, thus the cells could start and guide the
fermentation.
A future perspective could be the optimization of the kinetic of cell
release for an effective modulation of the acidification of meat, as well
as to assure the presence of the released microorganism in the product
up to consumption.

Nitrites – – −3.29
Acknowledgments
Nitrates – – _
NaCl – – −3.97
Temperature – – – This paper was supported by the Italian Ministry of Education and
Lactose – 5.77 4.71 Research (MIUR) through the project PON-01_1409 “Process and
Pepper – 11.72 10.62 product innovations aimed at increasing food safety and at diversifying
R2ad – 0.966 0.9520
pork-based products (SAFEMEAT)”.
 M.R. Corbo et al. / Meat Science 111 (2016) 198–203
References
Anal, A.K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for in-
dustrial applications and targeted delivery. Trends in Food Science & Technology, 18,
240–251.
Bacus, J. (1984). Utilization of microorganisms in meat processing. A handbook for meat plant
operators. New York: John Wiley & Sons, Inc.
Bevilacqua, A., Corbo, M.R., & Sinigaglia, M. (2011). Shelf life of alginate beads containing
lactobacilli and bifidobacteria: Characterisation of microspheres containing Lactoba-
cillus delbrueckii subsp. bulgaricus. International Journal of Food Science and
Technology, 46, 2212–2217.
Box, G.E.P., Hunter, J.S., & Hunter, J.S. (2005). Statistics for experimenters. Design, innovation
and discovery (2nd ed.). New York: John Wiley & Sons, Inc.
Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic living cells:
From laboratory scale to industrial applications. Journal of Food Engineering, 104,
467–483.
Cardenas, F.C., Giannuzzi, L., & Zaritzky, N.E. (2007). Modelling microbial growth in meat
broth with added lactic acid under refrigerated storage. International Journal of Food
Science and Technology, 42, 175–184.
Champagne, C.P., & Fustier, P. (2007). Microencapsulation for the improved delivery of
bioactive compounds into foods. Current Opinion in Biotechnology, 18, 184–190.
Chávarri, M., Marañón, I., Ares, R., Ibáñez, F.C., Marzo, F., & Villarán, M. (2010). Microen-
capsulation of a probiotic and prebiotic in alginate-chitosan capsules improves
survival in simulated gastro-intestinal conditions. International Journal of Food
Microbiology, 142, 185–189.
Corbo, M.R., Bevilacqua, A., Gallo, M., Speranza, B., & Sinigaglia, M. (2013). Immobilization
and microencapsulation of Lactobacillus plantarum: Performances and in vivo applica-
tions. Innovative Food Science & Emerging Technologies, 18, 196–201.
Dianawati, D., Mishra, V., & Shah, N.P. (2013). Stability of microencapsulated Lactobacillus
acidophilus and Lactococcus lactis ssp. cremoris during storage at room temperature at
low aw. Food Research International, 50, 259–265.
203
Fritzen-Freire, C.B., Prudêncio, E.S., Amboni, R.D., Pinto, S.S., Negrão-Murakami, A.N., &
Murakami, F.S. (2012). Microencapsulation of bifidobacteria by spray drying in the
presence of prebiotics. Food Research International, 45, 306–312.
Gallo, M., Bevilacqua, A., Speranza, B., Sinigaglia, M., & Corbo, M.R. (2014). Alginate beads
and apple pieces as carriers for Saccharomyces cerevisiae var. boulardii, as representa-
tive of yeast functional starter cultures. International Journal of Food Science and
Technology, 49, 2092–2100.
Kailasapathy, K. (2002). Microencapsulation of probiotic bacteria: Technology and poten-
tial applications. Current Issues in Intestinal Microbiology, 3, 39–48.
Khali, A.H., & Mansour, E.H. (1998). Alginate encapsulated bifidobacteria survival in may-
onnaise. Journal of Food Science, 63, 702–705.
Lee, J.S., Cha, D.S., & Park, H.J. (2004). Survival of freeze-dried Lactobacillus bulgaricus KFRI
673 in chitosan-coated calcium alginate microparticles. Journal of Agricultural and
Food Chemistry, 52, 7300–7305.
Muthukumarasamy, P., & Holley, R.A. (2006). Microbiological and sensory quality of dry
fermented sausages containing alginate-microencapsulated Lactobacillus reuteri.
International Journal of Food Microbiology, 111, 164–169.
Nualkaekul, S., Lenton, D., Cook, M.T., Khutoryanskiy, V.V., & Charalampopoulos, D.
(2012). Chitosan coated alginate beads for the survival of microencapsulated
Lactobacillus plantarum in pomegranate juice. Carbohydrate Polymers, 90, 1281–1287.
Özer, B., Kirmaci, H.A., Şenel, E., Atamer, M., & Hayaloğlu, A. (2009). Improving the viabil-
ity of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-
brined cheese by microencapsulation. International Dairy Journal, 19, 22–29.
Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein-based mi-
crocapsules and survival in simulated gastrointestinal conditions and in yoghurt.
International Dairy Journal, 14, 505–515.
Prevost, H., & Divies, C. (1992). Cream fermentation by a mixed culture of lactococci
entrapped in two-layer calcium alginate gel beads. Biotechnology Letters, 14, 583–588.
Sabikhi, L., Babu, R., Thompkinson, D.K., & Kapila, S. (2010). Resistance of microencapsu-
lated Lactobacillus acidophilus LA1 to processing treatments and simulated gut condi-
tions. Food and Bioprocess Technology, 3, 586–593.