1088 International Journal of Food Science and Technology 2020, 55, 1088–1096

Original article
Probiotication of cooked sausages employing agroindustrial
coproducts as prebiotic co-encapsulant in ionotropic
alginate–pectin gels

an-Mart
ınez,1 Alfonso Totosaus1

Luis P. Barrag
& Mar
ıa de Lourdes P
erez-Chabela2*
1 Food Science Laboratory and Pilot Plant, Tecnologico Estudios Superiores Ecatepec, Av. Tecnologico esq. Av. Central s/n, Ecatepec 55210,
Mexico
2 Biotechnology Department, Universidad Aut
onoma Metropolitana Iztapalapa, Av. San Rafael Atlixco 86, Mexico City 09340, Mexico
(Received 29 March 2019; Accepted in revised form 4 June 2019)

Summary Cooked sausages were formulated inoculating alginate–pectin microcapsules containing agroindustrial
coproducts, cactus pear peel flour or apple marc flour, seeking to enhance the nutritional value of cooked
meat products. The microcapsules increased total moisture (from 66 to 75% in average), but water was
not being physically retained since higher expressible moisture values in inoculated samples were observed
(20% as compared to 15% in control). Inoculated samples presented higher lactic acid bacteria popula-
tions, since in addition to the thermotolerant capacity of the bacteria, encapsulation added a protective
barrier for the bacteria to survive. Higher lactic acid bacteria counts were reflected in fewer coliforms in
inoculated samples (<0.001 log CFU after 15 days of storage), with no detrimental effect on texture. Nat-
ural antioxidants present in agroindustrial coproducts decreased the oxidative rancidity of lipids for stor-
age. The results imply that agroindustrial coproducts are a good alternative to formulate symbiotic
functional ingredients that can be employed to improve the nutritional properties of nondairy thermal
processed food products, like cooked emulsified meat products.
Keywords Agroindustrial coproducts, microencapsulation, prebiotics, probiotic, sausages, symbiotic.

Probiotic strains that will be employed in meat

Introduction
Probiotics, such as lactic acid bacteria, are widely
employed in fermented dairy products and the strong
evidence of their health benefits on humans and ani-
mals has led to modern, forward-looking research to
develop their applications in nondairy foods. Factors
that affect microorganism behaviour and robustness in
the different food environments, and which must be
considered to ensure probiotic performance, can be
categorised into intrinsic and extrinsic. Intrinsic factors
are related to the type of culture selected, growth
stage, subcellular injuries by heat or osmotic stress and
the careful selection of strains with highest innate tech-
nological behaviour. Extrinsic factors include composi-
tion of food matrices, pH value, oxygen level, food
manufacturing conditions and storage time, use of cell
protectants, strain adaptation to a sublethal dose of a
specific physical or chemical stress, genetic manipula-
tion, the inclusion of probiotics in edible films or their
microencapsulation (De Prisco & Mauriello, 2016).
*Correspondent: E-mail: lpch@xanum.uam.mx.
products must have the ability to ferment the least
amount of carbohydrates in meat batter and tolerate
specific detrimental factors such as added sodium chlo-
ride and nitrate, for the purpose of being strong com-
petitors against natural meat microflora, and finally
survive in high numbers at the time of consumption to
exert beneficial effects on the host (Rouhi et al., 2013).
Nonetheless, unlike dairy products where starter or
probiotic cultures are always added to milk after pas-
teurisation, cooked meat products are heated to a core
temperature of 70–72 °C, where free microorganisms
are not able to survive. This process is necessary to
develop textural characteristics and destroy microbiota
in order to increase shelf life (Cavalheiro et al., 2015).
The survival of lactic acid bacteria under these particu-
lar conditions can be achieved with a protective cover.
Meat products are prone to contamination by
pathogenic microorganism, resulting in outbreaks
caused by contamination. In addition, consumers are
more aware of the importance of food safety, demand-
ing higher quality and natural no-additives added meat

doi:10.1111/ijfs.14259
© 2019 Institute of Food Science and Technology

foods. Antimicrobial agents as organic acids (like lac-
tic, benzoic and sorbic) are GRAS additives commonly
employed in meat industry, but with the limitation of
a potential negative impact on flavour and colour. In
order to reduce additives in meat products, lactic acid
bacteria can be employed as a natural biopreservative
agent. Lactic acid bacteria produce a variety of
compounds such as organic acids (lactic acid, acetic
acid), diacetyl, hydrogen peroxide, and bacteriocins or
bactericidal proteins all of which have antibacterial or
bacteriostatic properties. Bacteriocins are natural
antimicrobial preservatives that, in conjunction with
other metabolites, extend shelf life and inhibit the
growth of pathogenic organisms, exerting a positive
effect on food taste, smell, colour and texture
(Woraprayote et al., 2016; Zarour et al., 2017).
Encapsulants are nontoxic, stable and easily avail-
able materials, with special properties in terms of cost,
transferability, biological decomposition as well as
physical properties such as microbial growth possibil-
ity and solubility, and are intended to extend the via-
bility of cells and improve their potential effect by
entrapment in the matrix (Abress & Nateghi, 2015).
Prebiotics can selectively enhance the growth and/or
metabolism of probiotics in the intestine, but another
important function from a technological point of view
is that prebiotics could affect the viability of probiotics
for storage, specifically to improve the growth of pro-
biotic strains inoculated into meat products, on a kind
of ‘synbiotic’ basis (Rouhi et al., 2013). Co-encapsula-
tion offers the potential to increase the viability of
encapsulated bacteria, the effect of adding complemen-
tary prebiotics enhancing the efficacy of functional
foods by synergy between prebiotic and probiotic
ingredients (Iyer & Kailasapathy, 2005).
The objective of this work was to analyse the effects
of different agroindustrial coproducts, cactus pear peel
flour or apple marc flour, as prebiotic co-encapsulants
in ionotropic microencapsulation of thermotolerant
probiotic lactic acid bacteria, Enterococcus faecium
UAM1 or Pediococcus pentosaceus UAM2, on mois-
ture, texture, oxidative rancidity and lactic acid bacte-
ria growth on inoculated cooked sausages.
Materials and methods
Agroindustrial coproduct flour and ionotropic
encapsulation
Cactus pear (Opuntia ficus-indica L.) peels were recov-
ered from local fresh fruit establishments in Mexico
City for the 2017 season (May to August). The cactus
pear peels were washed with tap water, cut into
2 9 2 cm pieces and dried in a Weston model 74–
1001-w food dehydrator (Weston, Southern Pines) at
60 °C for approximately 24 h.
© 2019 Institute of Food Science and Technology

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ınez et al.
Probiotication of cooked sausages L. P. Barraga 1089
Marc from native Mexican apple (Malus domestica
var. rayada) was collected as coproduct from an apple
cider manufacturing facility (Bodegas Delicia,
Zacatl
an, Puebla) for the 2017 production period (July
to October). The apple marc was dried at 70 °C in the
same dehydrator as previously described for approxi-
mately 36 h.
Dried samples were ground in a grain mill and
sieved consecutively in No. 100, 80, 50 and 20 sieves
to obtain a regular and homogeneous powder named
flour. Different collected lots were mixed to obtain a
single batch. Cactus pear peel flour and apple marc
flour were stored in hermetic containers until use.
Two strains of lactic acid bacteria reported as ther-
motolerant and probiotic (Hern
andez-Alc
antara et al.,
2018), namely E. faecium UAM1 and P. pentosaceus
UAM2, were employed. The lactic acid bacteria strains
were reactivated in MRS broth at 37 °C for 24 h until
obtaining an optical density close to one (k = 600 nm),
equivalent to approximately 108 CFU mL
1. The cell
suspension was centrifuged at 2000 g for 15 min to
obtain a cellular pellet of each strain. Microencapsula-
tion of bacteria strains was carried out adapting the
technique of Homayouni et al. (2007). Each cellular
pellet was resuspended in 100 mL of a 0.4 M CaCO3
solution containing 2.0% (w/v) of alginate (PROTA-
NAL SF 120 sodium alginate, FMC Biopolymers,
Philadelphia, PA, USA) and pectin (GRINDSTED
LA 110 pectin; Danisco, Madison, WI, USA) in a
50:50 proportion plus 1% (w/v) of cactus pear peel
flour, apple marc flour or inulin as control, as prebi-
otic co-encapsulant. The oily phase was prepared with
20 mL of MazolaÒ maize oil (ACH Food Co., Mexico
City, Mexico) and 2.5 mL of Tween-80 (Sigma-
Aldrich, St. Louis, MO, USA). An emulsion was
formed dispersing both phases with magnetic stirring
at high speed for 10 min (ca. 400 r.p.m.), until the
addition of 40 mL of maize oil with 2 mL of acetic
acid (0.35 M) to allow the formation of micro-gel
beads. After 20 min, the microcapsules were removed
from the aqueous phase and washed twice with
10 mM sodium phosphate buffer solution, pH 7.2. The
microcapsules were stored in hermetic containers at
4 °C to allow full hardening for at least 24 h.
Sausage elaboration
Lean pork and lard (pork backfat) were purchased in
local abattoirs, removing visible fat and connective tis-
sue. Meat (50% w/w) was ground through a 0.42-cm
plate in a meat grinder and mixed with salt (2% w/w),
commercial phosphate mixture (FABPSA, M
exico
City, M
exico, 0.8% w/w) and curing salt (0.3% w/w
containing 0.5% of NaNO2, equivalent to 0.0015%
NaNO2 of residual nitrite after 15 days of storage, in
agree with Ref. (Baz
an-Lugo et al., 2011). Moreover,
International Journal of Food Science and Technology 2020
1090
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ınez et al.
Probiotication of cooked sausages L. P. Barraga
it is suggested to add the reference Baz

an-Lugo et al.,
with half of the total ice for two min in a Chef Prep
70610 Food Processor (Hamilton Beach, Glen Allen,
VA, USA). Frozen lard (20%) was added and emulsi-
fied for 2–3 min. The rest of the ice was added and
emulsified for 2–3 min, adding wheat flour (5% w/w)
until total ingredient incorporation, maintaining the
batter temperature at 12  2 °C. Finally, microcap-
sules (5% w/w) were incorporated. The batters were
stuffed into 20-mm-diameter cellulose casing and
cooked in a water bath until reaching an internal tem-
perature of 70  2 °C (about 15 min), then cooled in
an ice bath, vacuum-packed in CryovacÒ B2540 bags
(Sealed Air, Monterrey, Mexico) and stored at 4 °C
until subsequent analysis at 1, 15 and 30 days of stor-
age. For each treatment, a total of total of three
batches (1 kg each one) were manufactured.
Total moisture, expressible moisture and recooking
stability
Moisture content was quantified using AOAC Official
Test Method No. 950.46 (AOAC, 1999). Two grams
of samples was placed in an aluminium capsule at con-
stant weight and heated in an oven at 110 °C for 12 h.
The samples were then removed, and the percentage of
moisture was calculated based on weight difference.
Expressible moisture was determined adapting the
methodology reported by Jauregui et al. (1981). Three
pieces of Whatman #4 filter paper were weighted,
folded into a thimble shape with 2  0.3 g of ground
meat batter sample and centrifuged at 3000 g for
20 min at 4 °C. Expressible moisture was reported as
the percentage of weight lost from the original weight
of the sample.
Recooking stability was determined by modifying
Haq et al. (1972) methodology. Cooked sausages (ca.
30 g) were heated in 100 mL water at 70 °C for
30 min; cooking stability was reported as the percent-
age of the weight difference between the samples
before and after recooking.
Instrumental colour
Instrumental colour of the internal part of the samples
was determined on the CIE-Lab scale employing the
Color Analysis application for Android O.S. (Research
Lab Tools, S~ ao Paulo, Brazil). CIE Standard: Illumi-
nant = D65, corresponding to the average midday
light (comprising both direct sunlight and the light dif-
fused by a clear sky, also called a daylight illuminant);
and standard observer angle function = 2 °. Results
are the average of four readings rotating each sample
by 90 °. From the CIE-Lab values, the hue angle (H)
and saturation index (S) were calculated as described
by Little (1975), according to:
International Journal of Food Science and Technology 2020
b
Hue angle (H) ¼ Tan
ð1Þ
a
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Satuartion index(S) ¼ a2 þ b2 ð2Þ
The total colour difference (DE) in inoculated sam-
ples, considering the control sample as reference (Cava
et al., 2012), was calculated as:
Colour differenceðDEÞ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
¼ ðLControl
L Þ2 þ ðaControl
a Þ2 þ ðbControl
b Þ2
ð3Þ
Lactic acid bacteria and coliform counts and pH
For the microbiological counts, 10 g of sausage sample
was homogenised aseptically in a sterile Waring blen-
der jar with 90 mL of saline solution (0.85%, w/v).
After blending, pertinent decimal dilutions were pour-
plated (0.1 mL) onto MRS agar for lactic acid bacteria
(incubated at 37 °C for 24 h) or Violet Red Bile Glu-
cose agar for coliforms (incubated at 37 °C for 24 h),
converting microbial counts to logarithms of colony-
forming units (CFU) per gram (log CFU/g).
The pH was determined according to Landvogt’s
(1991) recommendations for measuring pH in meat
products. Ten grams of sample was homogenised in
100 mL of 5% (w/v) NaCl solution and pH measured
with a potentiometer (Beckman Instruments, Palo
Alto, CA, USA).
Oxidative rancidity
Oxidative rancidity in sausage samples was determined
using the methodology of Zipser & Watts (1962). Ten
grams of ground sample was mixed with 49 mL of dis-
tilled water at 50 °C, adding one mL of sulphanil-
amide–HCl solution (0.5% and 20%, respectively, v/
v). Subsequently, the sample was transferred to a 500-
mL Erlenmeyer flask containing 48 mL of distilled
water at 50 °C and 2 mL of HCl solution (50% v/v),
plus 2 drops of silicone-based antifoam. The contents
of the flask were distilled for 10–15 minutes or until
obtaining 50 mL of distillate. An aliquot of 5 mL was
taken and mixed with 5 mL of thiobarbituric acid
solution (0.02 M in glacial acetic acid 90%). Samples
were placed in boiling water for 35 min and cooled,
and the absorbance was measured at 538 nm. The con-
centration of malondialdehyde (mg kg
1 of sample)
was calculated by extrapolating the absorbance against
a 1,1,3,3-tetraethoxypropane (3 9 103 g L
1) solution.
Textural profile analysis
Sausage samples from each treatment were cut into
2 cm lengths to perform a texture profile analysis
© 2019 Institute of Food Science and Technology

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ınez et al.
Probiotication of cooked sausages L. P. Barraga 1091

(TPA), compressing axially in two consecutive cycles
(50% original height) with a 40-mm-diameter acrylic
probe at a cross-head speed of one mm/s, with a 5-s
waiting period, in a LFRA 4500 Texture Analyzer
(Brookfield Engineering, Middleboro, MA, USA).
Textural parameters were calculated from the force–
time curves as follows: hardness (force necessary to
attain a given deformation, maximum force), cohesive-
ness (strength of the internal bonds making up the
body of the product) and springiness (the extent to
which a product returns to its original shape when
compressed) (Szczesniak, 1963; Bourne, 1978). Resili-
ence (energy absorbed by the sample during compres-
sion and then released, during the first compression)
was determined from force–deformation curves by
measuring the area enclosed by the hysteresis loop,
that is, energy stored in the sample that allows the
recovery to some extent of its original shape (Voisey
et al., 1975). Results are the mean of at least five
reproducible runs for each treatment per batch.
Experimental design and data analysis
In order to determine the effect of agroindustrial
coproducts such as prebiotic co-encapsulant in the co-
encapsulation of lactic acid bacteria into a alginate:
pectin gel matrix on cooked sausage properties during
30 days of storage, the proposed model was:
yij ¼ l þ ai þ bj þ eij
where yij represents the sausage total moisture,
expressible moisture, recooking stability, colour,
microbiological counts, pH, oxidative rancidity and
instrumental texture for the ith treatment at the jth
day of storage; ai and bj are the main effects of treat-
ment and storage time; and
ij represents the residual
error terms, assumed to be normally distributed, with zero
mean and variance r2 (Der & Everitt, 2008). Data analy-
sis was carried out using SAS statistical software v. 8.0
(SAS Institute, Cary, NC, USA) with PROC GLM proce-
dure. Significant difference among means was determined
by Duncan’s means test (P = 0.05) in the same software.
Results and discussion
Moisture, expressible moisture, cooking stability and yield
Parameters related to water properties in cooked sau-
sages inoculated with encapsulated lactic acid bacteria
are depicted in Table 1. Total moisture content was
significantly (P < 0.05) higher for both samples inocu-
lated with cactus pear peel flour and apple marc with
E. faecium. Inulin containing treatments for both
microorganisms presented low total moisture values,
but was lower in control samples. Moisture decreased
significantly (P < 0.05) with storage time.
Cactus pear peel flour with E. faecium obtained sig-
nificantly (P < 0.05) higher values for expressible mois-
ture, followed by the apple marc with E. faecium
treatment. Lower expressible moisture values were
observed in control samples. Expressible moisture
decreased significantly (P < 0.05) during storage time
(Table 1).
For recooking stability, both cactus pear peel flour
and apple marc with E. faecium obtained significantly
(P < 0.05) higher values than the control samples. The
lowest cooking stability was observed in inulin con-
taining treatments for both microorganisms.

Table 1 Total moisture, expressible moisture and recooking stability for the sausages formulated with encapsulated lactic acid
bacteria (E.f.: Enterococcus faecium, P.p.: Pediococcus pentosaceus) with agroindustrial coproducts as prebiotic co-encapsulant

Storage
time Apple marc Apple marc Cactus pear peel Cactus pear peel
(days) Control Inulin + E. f. Inulin + P. p. flour+ E. f. flour + P. p. flour + E. f. flour + P. p.

Total moisture (%)

1 66.19  0.08 e, A 74.66  0.47 c, A 73.80  0.32 d, A 80.91  0.14 a, A 78.54  0.42 b, A 80.19  0.16 a, A 80.14  0.40 b, A
15 66.64  0.41 e, B 72.63  0.07 c, B 72.83  2.56 d, B 75.22  0.13 a, B 74.05  0.82 b, B 72.19  0.77 a, B 76.60  0.33 b, B
30 64.80  1.01 e, C 71.43  0.72 c, C 64.90  0.93 d, C 47.25  0.08 a, C 73.42  0.69 b, C 71.06  2.12 a, C 73.26  1.39 b, C
Expressible moisture (%)
1 16.30  0.21 f, A 16.01  0.89 d, A 22.79  0.11 e, A 25.56  0.62 b, A 22.77  0.38 c, A 36.85  0.06 a, A 22.40  0.29 c, A
15 14.07  0.25 f, B 14.86  0.05 d, B 13.10  0.03 e, B 21.01  0.38 b, B 21.96  0.19 c, B 21.76  0.43 a, B 21.60  0.72 c, B
30 13.20  0.62 f, C 14.05  0.32 d, C 12.73  0.07 e, C 19.88  0.21 b, C 19.04  0.50 c, C 16.48  0.20 a, C 19.45  0.16 c, C
Recooking stability (%)
1 92.46  0.30 e, C 91.78  0.87 c, C 93.56  0.64 d, C 97.13  0.14 a, C 95.66  0.63 b, C 98.51  0.21 a, C 96.43  0.07 b, C
15 91.75  0.68 e, B 99.56  0.44 c, B 95.15  0.70 d, B 98.72  0.01 a, B 95.98  0.14 b, B 96.53  0.14 a, B 95.86  1.13 b, B
30 97.04  0.27 e, A 95.01  0.05 c, A 98.00  0.32 d, A 94.97  0.42 a, A 98.55  0.24 b, A 98.06  0.92 a, A 96.62  0.30 b, A

a, b, c. . . Means with same letter are not significantly (P > 0.05) different for treatment.
A, B, C. . . Means with same letter are not significantly (P > 0.05) different for storage time.

© 2019 Institute of Food Science and Technology International Journal of Food Science and Technology 2020
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Recooking stability increased significantly (P < 0.05)
with storage time (Table 1).
The inoculation of alginate–pectin microcapsules
with cactus pear peel flour or apple marc flour
containing lactic acid bacteria enhanced water con-
tent within meat protein matrix, although this
moisture cannot be physically retained, which is
reflected in high expressible moisture test values.
Nonetheless, for the recooking process of the sam-
ples, fibres in flours improved water entrapment,
increasing water retention. Water seems to be sta-
bilised by the inoculated microcapsules with both
cactus pear peel flour and apple marc flour, also
during the storage period. In chicken sausages,
apple pomace fibre incorporation resulted in less
cooking loss (Choi et al., 2016). Similarly, cactus
pear peel fibres contained the greatest amount of
cellulose and hemicellulose and lesser amounts of
lignin (El Kossori et al., 1998), fibres that enhance
water retention in cooked meat products (Fern
an-
dez-L
opez et al., 2004).
Instrumental colour
Table 2 shows the effect of inoculated microencapsu-
lated lactic acid bacteria on sausage colour. Luminos-
ity was significantly (P < 0.05) higher for cactus pear
peel flour treatments with P. pentosaceus; and lower
values, with darker coloration, were observed in the
control samples. Sample luminosity decreased signifi-
cantly (P < 0.05) with storage time. Hue angle was
significantly (P < 0.05) higher for samples containing
cactus pear peel flour with P. pentosaceus microcap-
sules, followed by the apple marc flour with P. pen-
tosaceus treatments; lower values for this colour
parameter were found in the control samples. Hue
angle significantly (P < 0.05) decreased during storage.
Saturation index values were significantly (P < 0.05)
higher for cactus pear peel flour with P. pentosaceus
treatment, followed by the cactus pear peel flour with
E. faecium microcapsules treatment; lower values were
observed in the control samples. In addition, the satu-
ration index decreased significantly (P < 0.05) as well
as storage time. Colour difference was significantly
(P < 0.05) higher for cactus pear peel flour with
P. pentosaceus microcapsules. The lowest colour differ-
ence was detected in apple marc flour microcapsules
with P. pentosaceus. Colour difference significantly
(P < 0.05) increases with storage time.
Higher moisture content showed lighter coloration
in meat batters (Youssef & Barbut, 2011). Treatments
with microcapsules presented higher moisture content,
explaining the brighter colour. In the same way, col-
oration of the samples became darker during storage,
in association with the lower moisture content. Cactus
pear peel flour treatments increased both colour tone
(H) and colour intensity of the samples, probably due
to the presence of moisture content and peel pigments.
Both colour tonality and intensity became stable with
storage. During the first 15 days of storage, colour dif-
ferences between control and inoculated samples
showed a moderate degree of dissimilarity (DE < 6.0),

Table 2 Instrumental colour for the sausages formulated with encapsulated lactic acid bacteria (E.f.: Enterococcus faecium, P.p.:
Pediococcus pentosaceus) with agroindustrial coproducts as prebiotic co-encapsulant

Storage
time Apple marc Apple marc Cactus pear peel Cactus pear peel
(Days) Control Inulin + E. f. Inulin + P. p. flour + E. f. flour + P. p. flour + E. f. flour + P. p.

Luminosity L*
1 77.05  0.93 f, A 81.95  0.49 e, A 74.25  0.16 d, A 81.33  0.58 c, A 65.98  0.12 e, A 80.91  0.65 b, A 86.95  0.16 a, A
15 62.65  0.49 f, B 71.69  0.09 e, B 71.11  0.32 d, B 69.85  0.82 c, B 78.05  0.71 e, B 81.76  0.62 b, B 76.71  0.67 a, B
30 61.08  0.64 f, C 61.20  0.43 e, C 70.84  0.29 d, C 67.39  0.45 c, C 74.20  0.32 e, C 71.66  0.62 b, C 74.44  0.72 a, C
Hue angle (H)
1 1.358  0.09 e, A 1.291  0.04 d, A 1.247  0.10 c, A 1.142  0.05 d, A 1.263  0.01 b, A 1.403  0.08 d, A 1.323  0.02 a, A
15 1.015  0.04 e, A 1.138  0.09 d, A 1.219  0.03 c, A 1.138  0.02 d, A 1.112  0.06 b, A 0.774  0.02 d, A 1.277  0.01 a, A
30 0.978  0.06 e, B 0.667  0.03 d, B 0.919  0.02 c, B 0.552  0.02 d, B 1.098  0.07 b, B 0.693  0.04 d, B 1.178  0.02 a, B
Saturation index (S)
1 8.60  0.14 e, A 9.35  0.17 c, A 8.61  0.16 d, A 10.78  0.06 b, A 8.01  0.07 d, A 11.93  0.11 b, A 9.73  0.07 a, A
15 8.21  0.38 e, B 8.70  0.21 c, B 8.03  0.12 d, B 10.43  0.18 b, B 7.66  0.04 d, B 11.09  0.12 b, B 9.23  0.20 a, B
30 8.18  0.03 e, C 3.18  0.18 c, C 7.49  0.14 d, C 6.23  0.36 b, C 6.51  0.24 d, C 10.87  0.03 b, C 8.71  0.26 a, C
Colour difference (DE)
1 - 3.25  0.09 d, C 2.64  0.29 d, C 4.25  0.08 c, C 5.75  0.16 e, C 6.94  0.06 b, C 5.76  0.23 a, C
15 - 5.73  0.11 d, B 5.14  0.06 d, B 5.16  0.53 c, B 7.57  0.27 e, B 6.57  0.14 b, B 8.08  0.65 a, B
30 - 11.60  0.08 d, A 10.69  0.04 d, A 12.92  0.19 c, A 12.20  0.42 e, A 13.43  0.33 b, A 13.80  0.16 a, A

a, b, c. . . Means with same letter are not significantly (P > 0.05) different for treatment.
A, B, C. . . Means with same letter are not significantly (P > 0.05) different for storage time.

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but at 30 days, the colour difference was noticeable
(DE > 12.0) (Sal
akov

a, 2012).
Lactic acid bacteria, coliforms and pH
Lactic acid bacteria and coliform counts during stor-
age are listed in Table 3. Significantly (P < 0.05)
higher lactic acid bacteria counts were detected in sam-
ples with cactus pear peel flour microcapsules with
E. faecium. Lowest lactic acid bacteria counts were
detected in both apple marc flour with P. pentosaceus
or E. faecium samples, and as expected, the lower lac-
tic acid bacteria were detected in control samples. Lac-
tic acid bacteria increased significantly (P < 0.05) with
storage time. Inoculated samples resulted as well in
significantly (P < 0.05) lower coliforms counting.
Lower coliforms were detected in treatments contain-
ing cactus pear peel flour. Coliforms decreased signifi-
cantly (P < 0.05) with storage time.
Significantly (P < 0.05) lower pH values were
observed in samples inoculated with microcapsules
containing cactus pear peel flour with P. pentosaceus
or E. faecium; despite the higher pH in noninoculated
control samples, highest pH was observed when apple
marc flour was employed to microencapsulate both
microorganisms. Sausage pH decreased significantly
(P < 0.05) with storage time as a consequence of
microbial growth (Table 3).
In packed meat products like cooked sausages, inter-
actions between factors like nutrients and water
availability influence the growth of lactic acid bacteria.
Initially present microorganisms are affected by selec-
tion (based primarily on nutrient composition and
physicochemical parameters), and in meat products,
vacuum-packaging and salt content provoke a selective
pressure favouring lactic acid bacteria (Gram et al.,
2002). In meat emulsion, the fat in the system also
envelops the alginate microcapsules with the bacterial
cells as a coupled protection during thermal processing
(Manojlovi
c et al., 2010). Under the experimental con-
ditions employed, inoculated microencapsulated lactic
acid bacteria presented higher microbial counts at
15 days of storage, since the stationary phase in vac-
uum-packaged cooked meat emulsions is reached after
8–10 days of storage (Cayr
e et al., 2006).
This was reflected in two aspects. First, the higher
number of cells in samples containing microcapsules
and second the relatively low pH in samples contain-
ing microcapsules, compared to the control samples.
Cactus pear peel flour and apple marc flour promoted
microbial growth even more than samples containing
inulin, the prebiotic by definition. Diaz-Vela et al.
(2013) reported that in in vitro studies cactus pear flour
improved lactic acid bacteria metabolism (higher maxi-
mum acidification rate and carbohydrate consump-
tion), since although complex substrates like insoluble
fibre peel flour resulted in lower growth rate, the final
biomass was similar to glucose as a carbon source.
Immobilised cells present rapid fermentation rates
compared to free cells due to the available nutrients in

Table 3 Lactic acid bacteria and coliform count, pH and oxidative rancidity for the sausages formulated with encapsulated lactic
acid bacteria (E.f.: Enterococcus faecium, P.p.: Pediococcus pentosaceus) with agroindustrial coproducts as prebiotic co-encapsu-
lant

Storage
time Apple marc Apple marc Cactus pear peel Cactus pear peel
(Days) Control Inulin + E. f. Inulin + P. p. flour + E. f. flour + P. p. flour + E. f. flour + P. p.

LAB (log UFC/g)

1 2.72  0.00 g, A 4.50  0.03 c, A 4.50  0.03 d, A 4.71  0.01 e, A 4.50  0.03 f, A 4.71  0.01 a, A 4.57  0.01 b, A
15 6.63  0.03 g, B 6.87  0.01 c, B 6.79  0.01 d, B 6.56  0.02 e, B 6.51  0.04 f, B 7.60  0.00 a, B 7.05  0.01 b, B
30 4.05  0.05 g, C 7.49  0.01 c, C 7.11  0.01 d, C 7.02  0.02 e, C 6.75  0.01 f, C 7.83  0.02 a, C 7.49  0.00 b, C
Coliforms (log CFU/g)
1 3.78  0.01 a, A 3.63  0.01 e, A 3.72  0.01 d, A 3.70  0.01 c, A 3.85  0.01 b, A 3.56  0.02 f, A 3.57  0.01 g, A
15 5.15  0.01 a, C 0.00  0.00 e, C 0.00  0.00 d, C 0.00  0.00 c, C 0.00  0.00 b, C 0.00  0.00 f, C 0.00  0.00 g, C
30 5.65  0.01 a, B 0.00  0.00 e, B 0.00  0.00 d, B 0.00  0.00 c, B 0.00  0.00 b, B 0.00  0.00 f, B 0.00  0.00 g, B
pH
1 6.41  0.005 a, A 6.47  0.011 e, A 6.46  0.005 d, A 6.51  0.011 c, A 6.49  0.000 b, A 6.39  0.000 g, A 6.41  0.000 f, A
15 6.21  0.011 a, B 5.84  0.011 e, B 5.92  0.005 d, B 6.00  0.005 c, B 6.03  0.005 b, B 5.82  0.005 g, B 5.84  0.000 f, B
30 6.17  0.005 a, C 5.70  0.000 e, C 5.82  0.011 d, C 5.75  0.000 c, C 5.92  0.000 b, C 5.66  0.005 g, C 5.69  0.005 f, C
Oxidative rancidity (mg malondialdehyde kg
1)
1 0.254  0.002 a, C 0.249  0.001 c, C 0.254  0.001 b, C 0.247  0.002 d, C 0.251  0.002 e, C 0.249  0.002 g, C 0.246  0.002 f, C
15 0.491  0.003 a, B 0.429  0.002 c, B 0.443  0.001 b, B 0.414  0.001 d, B 0.415  0.001 e, B 0.389  0.001 g, B 0.405  0.001 f, B
30 0.566  0.003 a, A 0.490  0.001 c, A 0.505  0.001 b, A 0.480  0.001 d, A 0.478  0.001 e, A 0.465  0.002 g, A 0.463  0.004 f, A

a, b, c. . . Means with same letter are not significantly (P > 0.05) different for treatment.
A, B, C. . . Means with same letter are not significantly (P > 0.05) different for storage time.

© 2019 Institute of Food Science and Technology International Journal of Food Science and Technology 2020
1094
n-Mart
ınez et al.
Probiotication of cooked sausages L. P. Barraga
the protective environment of the alginate microcap-
sule (Manojlovi
c et al., 2010). Additionally, higher
moisture content in sausages provides a better environ-
ment for bacterial growth (Andr
es et al., 2006).
Antagonistic activities of lactic acid bacteria include
their ability to rapidly produce considerable acidic end
products with a concomitant pH reduction, besides
other metabolic products (hydrogen peroxide, diacetyl
and bacteriocins) that contribute to the overall
antibiosis and preservative potential (Lindgren &
Dobrogosz, 1990). Competition creates different types
of stress (nutrient limitation, acidification, atmosphere
and medium modification), and defence responses to
the hostile environment created by the competitors are
stress response and repair mechanisms (Andreevskaya
et al., 2018). Here, the thermotolerant capacity of the
employed strains allows them to withstand the encap-
sulation process and thermal treatment to survive and
become the dominant flora in inoculated cooked sau-
sages. Cactus pear peel flour has been used as func-
tional ingredient with prebiotic potential in cooked
meat products (Diaz-Vela et al., 2015).
Oxidative rancidity
As expected, lipid oxidative rancidity was significantly
(P < 0.05) higher in control samples without microcap-
sules. Samples containing cactus pear peel flour for
both microorganisms presented lower values, followed
by the apple marc treatments. Oxidative rancidity
increased significantly (P < 0.05) with storage time,
but to a lesser degree in samples with microcapsules
compared to control (Table 3).
Incorporation of fruit peels containing natural
antioxidant compounds decreases the lipid oxidation
in cooked meat products. Apple pomace or marc is a
source of polyphenols, derived from the cider-making
industry, with appreciable antioxidant activity (Di~
neiro
Garc
ıa et al., 2009). In cactus pear peel, antioxidants
such as total phenolics, tannin and flavonoid contents
have a higher antioxidant capacity (Cardador-
Mart
ınez et al., 2011). In cooked sausages, Diaz-Vela
et al. (2015) reported a decrease in oxidative rancidity
of cooked sausages when employing cactus pear peel
as functional fibre when thermotolerant lactic acid
bacteria were inoculated.
Textural profile analysis
Sausages inoculated with microcapsules containing
cactus pear peel flour with P. pentosaceus were signifi-
cantly (P < 0.05) harder than with the rest of the treat-
ments. A softer texture was observed in control
samples. Sausages became significantly (P < 0.05)
harder during storage. Cohesiveness of the control
samples presented significantly (P < 0.05) higher
International Journal of Food Science and Technology 2020
values, followed by both inulin containing microcap-
sule treatments. Sausages containing cactus pear peel
flour microcapsules with P. pentosaceus or E. faecium
were significantly (P < 0.05) less cohesive. Cohesive-
ness of the samples decreased significantly (P < 0.05)
with storage time. Control noninoculated samples pre-
sented significantly (P < 0.05) higher springiness val-
ues. Lower springiness was observed in inulin and
cactus pear peel flour containing microcapsules with
P. pentosaceus or E. faecium, with the lower values for
P. pentosaceus treatments. Springiness significantly
(P < 0.05) increased with storage time. Sausages resili-
ence was significantly (P < 0.05) higher for control
samples, with the lower values observed in cactus pear
peel flour with P. pentosaceus treatment. Sausage resi-
lience increased significantly (P < 0.05) with storage
time (Table 4).
Inclusion of alginate–pectin microcapsules resulted
in a harder but less cohesive and less ductile texture
(lower elasticity and flexibility). The main effect of
microcapsules can be attributed to the fibre contribu-
tion, plus water retention enhancement. Fibre incorpo-
ration resulted in less cooking loss and an increase in
sausage hardness (Choi et al., 2016). Dietary fibre is a
source of valuable pectin and dietary fibre from apple
pomace that are successfully transferred to meat prod-
ucts, lowering hardness and springiness (Jung et al.,
2015). Fibre incorporation increases the stability of
emulsions because water can be bound to insoluble
polysaccharides forming an insoluble three-dimen-
sional net increasing the consistency of the continuous
phase of meat emulsions, improving water and fat
retention (Cava et al., 2012).
Microencapsulation of probiotics is an option for
formulation of meat products to ensure that a desired
level of probiotic organisms is maintained in the final
product at consumption (L€ ucke, 2000). Microcapsules
could be used to protect bacterial viability only if the
microcapsulates were without effect on product sen-
sory quality. The alginate beads resembled, in terms of
both size and colour, discrete fat particles in the sau-
sage matrix were undetected (Rouhi et al., 2013). In
dry fermented sausages, small and irregularly shaped
microcapsules were imperceptible for panellists
(Muthukumarasamy & Holley, 2006). Since factors
related to cooked sausages processing (thermal pro-
cessing and vacuum-packaging under refrigeration)
decrease lactic acid bacteria metabolisms, the develop-
ment of higher quantities of lactic acid could affect
sensory acceptation of the product, nor other metabo-
lites against pathogenic microorganisms.
Conclusion
Microencapsulation of thermotolerant probiotic
microorganisms employing agroindustrial coproducts
© 2019 Institute of Food Science and Technology

n-Mart
ınez et al.
Probiotication of cooked sausages L. P. Barraga 1095

Table 4 Textural profile analysis for the sausages formulated with encapsulated lactic acid bacteria (E.f.: Enterococcus faecium,
P.p.: Pediococcus pentosaceus) with agroindustrial coproducts as prebiotic co-encapsulant

Storage
time Apple marc Apple marc Cactus pear peel Cactus pear peel
(Days) Control Inulin + E. f. Inulin + P. p. flour + E. f. flour + P. p. flour + E. f. flour + P. p.

Hardness (N)
1 22.29  2.31 e, C 17.40  0.11 f, C 17.48  0.42 g, C 22.71  0.70 d, C 20.01  0.13 b, C 21.70  1.20 c, C 34.10  0.32 a, C
15 25.77  0.85 e, B 22.00  0.32 f, B 19.09  0.72 g, B 27.51  0.70 d, B 25.75  0.93 b, B 28.18  0.68 c, B 35.15  0.93 a, B
30 27.10  0.21 e, A 27.25  0.27 f, A 21.15  0.06 g, A 27.29  1.16 d, A 29.00  0.43 b, A 31.03  2.09 c, A 36.15  0.48 a, A
Cohesiveness
1 0.837  0.004 a, A 0.783  0.004 b, A 0.755  0.002 c, A 0.736  0.004 d, A 0.750  0.003 d, A 0.738  0.007 e, A 0.716  0.006 f, A
15 0.815  0.001 a, B 0.770  0.000 b, B 0.746  0.009 c, B 0.720  0.010 d, B 0.736  0.002 d, B 0.721  0.009 e, B 0.684  0.035 f, B
30 0.791  0.011 a, C 0.760  0.002 b, C 0.734  0.010 c, C 0.714  0.016 d, C 0.722  0.005 d, C 0.713  0.001 e, C 0.617  0.006 f, C
Springiness (mm)
1 0.846  0.007 a, C 0.853  0.001 c, C 0.839  0.005 d, C 0.844  0.006 b, C 0.841  0.002 b, C 0.813  0.013 c, C 0.801  0.001 d, C
15 0.884  0.000 a, B 0.868  0.000 c, B 0.869  0.005 d, B 0.852  0.013 b, B 0.869  0.029 b, B 0.878  0.002 c, B 0.817  0.014 d, B
30 0.894  0.002 a, A 0.891  0.025 c, A 0.873  0.011 d, A 0.879  0.001 b, A 0.889  0.001 b, A 0.884  0.002 c, A 0.858  0.004 d, A
Resilience (N mm)
1 110.35  2.24 a, C 149.45  0.60 d, C 108.44  1.10 c, C 117.13  0.80 c, C 109.45  1.08 b, C 120.03  1.08 b, C 104.09  1.05 e, C
15 178.08  0.75 a, B 103.24  0.76 d, B 122.75  0.75 c, B 130.89  0.47 c, B 143.70  1.15 b, B 116.32  0.23 b, B 143.36  2.88 e, B
30 203.07  1.24 a, A 122.64  0.37 d, A 133.41  1.02 c, A 129.04  0.61 c, A 122.64  1.24 b, A 134.95  0.29 b, A 136.26  1.13 e, A

a, b, c. . . Means with same letter are not significantly (P > 0.05) different for treatment.
A, B, C. . . Means with same letter are not significantly (P > 0.05) different for storage time.

such as cactus pear peel flour or apple marc flour is a
good alternative to improve the nutritional properties
of cooked meat products with minor changes in colour
and texture. Microcapsule inoculation resulted in a
softer but more cohesive texture. Prebiotic properties
of cactus pear peel flour and apple marc flour pre-
sented a higher count of lactic acid bacteria, even
above inulin containing samples, inhibiting pathogens
like coliforms. The use of lactic acid bacteria encapsu-
lated with a prebiotic as symbiotic ingredient ensured
lower oxidative rancidity of fats. The use of agroindus-
trial coproducts as a source of prebiotic co-encapsu-
lant ingredient in ionotropic alginate–pectin gel
microencapsulation is an advantageous alternative to
nondairy food matrix probiotication.
Conflict of interest
The authors have declared no conflict of interest.
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