Food Chemistry 393 (2022) 133419

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Production of low-cholesterol butter with Lacticaseibacillus paracasei

immobilized in calcium-alginate beads
M.F.B. Teixeira a, S.P.M. Silva a, M.F.P. Domingos-Lopes a, b, R.J.B. Bessa c, d, J.A.M. Prates c,
H.J.D. Rosa a, C.C.G. Silva a, *
a
IITAA – Institute of Agricultural and Environmental Research and Technology, University of the Azores, Angra do Heroísmo, Portugal
b
CBA – Biotechnology Centre of Azores, University of the Azores, Angra do Heroísmo, Portugal
c
CIISA, Faculty of Veterinary Medicine, University of Lisbon, Lisboa, Portugal
d
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS)

A R T I C L E I N F O A B S T R A C T

Keywords: This study focused on the application of three strains of Lacticaseibacillus paracasei to assimilate cholesterol in
Cholesterol cream and butter. The strains were enclosed in calcium-alginate beads and incubated in cream at 30 ◦ C for 15 h.
Alginate Immobilization of lactobacilli cultures in calcium-alginate beads resulted in a 23% reduction in cholesterol (p <
Butter
0.05) in cream, whereas a negligible reduction was observed in cream fermented with free cells. Butter with a
Fermentation
Dairy cream
44% reduction in cholesterol was produced from fermented cream by L. paracasei L2A21K5 entrapped in alginate
Fatty acids beads. No significant (p > 0.05) changes in the fatty acid profile were observed in the low-cholesterol butter,
except for a slight but significant increase in n-3 fatty acids (p < 0.05). In addition, the indices of atherogenicity
and thrombogenicity were significantly reduced in the low-cholesterol butter (p < 0.05). Panelists rated the low-
cholesterol butter as good in appearance, consistency, and flavor.

1. Introduction
Cholesterol plays an important biochemical role in the human body
and can be synthesized in cells or ingested as part of the daily diet.
However, excessive blood cholesterol levels can lead to the development
of cardiovascular diseases, which are a major cause of mortality
worldwide (Mannarino, Ministrini, & Pirro, 2014; Seo & Choi, 2015).
Several reports indicate a dose-dependent association between low-
density lipoprotein cholesterol (LDL-C) and atherosclerosis (Mattiuzzi,
Sanchis-Gomar, & Lippi, 2020). In addition, recent reports have
confirmed that hypercholesterolemia is associated with a higher risk of
cardiovascular disease mortality (Mattiuzzi et al., 2020). Although the
role of dietary cholesterol remains controversial, dietary habits may be
one of the main causes of high blood cholesterol levels in people. Recent
studies reported that in overweight or obese individuals at high car­
diovascular risk, triglyceride and cholesterol levels were associated with
cardiovascular outcomes independent of other risk factors (Castañer
et al., 2020). For this reason, consumers are aware of the high choles­
terol content in foods and tend to choose foods with low cholesterol
content. In this regard, butter is considered unhealthy due to its high
content of saturated fatty acids and cholesterol (Silva, Silva, Prates,
Bessa, Rosa, & Rego, 2019).
Various methods have been investigated for the removal of choles­
terol from foods. These include nanofiltration (Allegre, Moulin, Gleize,
Pieroni, & Charbit, 2006), extraction by solid-phase or supercritical
fluids (Levit, Nazarova, Panarin, Korzhikova-Vlakh, & Tennikova, 2018;
Mohamed, Saldaña, Socantaype, & Kieckbusch, 2000) and complexation
with β-cyclodextrins (Alonso, Calvo, & Fontecha, 2019; Jia, Yang, Qi,
Chen, & Zhao, 2020; J. Kim, Jung, Ahn, & Kwak, 2006; Nguyen, Boakye,
Besong, Tomasula, & Alamu-Lumor, 2021). However, these methods
have several limitations, including the high cost of the process and a loss
of aroma, flavor, and consistency of the final product (Dias, Berbicz,
Pedrochi, Baesso, & Matioli, 2010; J. Kim et al., 2006; Y. Kim, Yoon,
Shin, Jo, Lee, & Kim, 2021). On the other hand, dairy products can serve
as carriers for probiotic cultures, including lactic acid bacteria (LAB).
Certain LAB not only have a positive effect on the fermentation process
of food, but can also have a positive effect on the health of the consumer.
These effects may include the production of various bioactive

* Corresponding author at: Instituto de Investigação e Tecnologias Agrárias e do Ambiente (IITAA), Universidade dos Açores, Rua Capitão João D’Avila, Angra do
Heroísmo, Portugal.
E-mail address: celia.cg.silva@uac.pt (C.C.G. Silva).

https://doi.org/10.1016/j.foodchem.2022.133419
Received 21 February 2022; Received in revised form 1 June 2022; Accepted 5 June 2022
Available online 6 June 2022
0308-8146/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.F.B. Teixeira et al.
compounds, and for this reason, some of these species can be used as
probiotics (Indira, Venkateswarulu, Peele, Bobby, & Krupanidhi, 2019;
Jurášková, Ribeiro, & Silva, 2022; Korcz, Kerényi, & Varga, 2018;
Linares et al., 2017). In addition, several authors reported that some
strains of lactobacilli can assimilate cholesterol during growth and this
assimilation can reduce the amount of cholesterol available for ab­
sorption from the intestine (Bosch, Fuentes, Audivert, Bonachera, Peiró,
& Cuñé, 2014; Hassan et al., 2019; Michael et al., 2017; Park et al., 2018;
Sivamaruthi, Kesika, & Chaiyasut, 2019). However, there are few
studies on the use of LAB to reduce cholesterol content in foods.
Recently, Kim et al. (2021) reported the use of LAB, which has choles­
terol assimilation ability, to reduce cholesterol content in cultured
butter. However, the method used required the addition of bile salts to
butter and the maximum cholesterol reduction was relatively low
(11%).
In a previous work, we investigated the ability of a series of LABs
isolated from artisanal Pico cheese to assimilate and reduce cholesterol
in the culture medium (Domingos-Lopes, Stanton, Ross, & Silva, 2020).
Three strains of Lacticaseibacillus paracasei (formerly Lactobacillus para­
casei) were found to have the ability to reduce cholesterol in the culture
medium. In the present study, we investigated the ability of these strains
entrapped in calcium-alginate beads to reduce cholesterol during the
fermentation of cream and produce a butter with low cholesterol con­
tent. In addition, the fatty acid profile and sensory properties of the low
cholesterol butter were also evaluated.
2. Materials & methods
2.1. Materials
Cream was obtained from cows’ milk from the University of the
Azores Farm (Chegalvorada). The milk was transported to the laboratory
and kept refrigerated at 4 ◦ C. The milk was subsequently (<24 h)
centrifuged at 3000 g at 4 ◦ C for 5 min, in order to separate the cream.
After this centrifugation, the cream was removed and kept refrigerated
(4 ◦ C) for use within 24 h or frozen (-20 ◦ C) until use. The cream used for
the experiments was standardized at 40% fat content. Before use, the
cream was pasteurized at 70 ◦ C for 20 min and immediately cooled in an
ice bath.
2.2. Bacterial cultures
Three strains of L. paracasei (strains L2A21K5, L3B1M2 and
L3B21R1) were previously isolated from artisanal Pico cheese and were
selected from the ability of reducing cholesterol in culture medium
(Domingos-Lopes et al., 2020). Each stock culture was stored at –70 ◦ C in
sterile de Mann Rogosa Sharpe (MRS) broth (Biokar, Beauvais, France)
supplemented with 30% (v/v) glycerol. The bacteria were activated
prior to testing by two sequential transfers in MRS broth supplemented
with 0.05% cysteine-HCl (Sigma-Aldrich, St. Louis, MO).
2.3. Immobilization of cells
Immobilization of bacterial cultures was performed under sterile
conditions with sterilized solutions (121 ◦ C for 15 min) according to
Prevost and Divies (1992) with minor modifications.
Activated cultures were inoculated (1%) into 50 mL of MRS broth
supplemented with 0.05% cysteine-HCl and incubated at 30 ◦ C for 12 h.
Tubes were then centrifuged at 4000×g for 10 min at 10 ◦ C (Eppendorf
5810 R, Hamburg, Germany). The cell pellet was washed with sterile
distilled water and centrifuged at 4000×g for 10 min. The cells were
then mixed with 40 mL of 2% sodium alginate solution (A3249, MW
10000–600,000g/mol, AppliChem, Panreac) previously sterilized
(100 ◦ C for 5 min) and used immediately. This mixture was placed in a
syringe (10 mL) and dropped into a sterile CaCl2 solution (0.05 M) with
the help of a needle (18 Gauge, inner diameter of 1.2 mm), to form
2
Food Chemistry 393 (2022) 133419
spheres. The spheres/beads were kept in the CaCl2 solution for 1 h and
then washed twice with sterile ultrapure water before use. Calcium-
alginate beads without the addition of cells were also prepared and
used as a control in cream fermentation.
2.4. Fermentation procedure and butter manufacture
Pasteurized cream (100 mL) was inoculated with the strains applied
directly to the cream (1%) or immobilized in calcium-alginate beads (40
mL). In both flasks the ratio of bacterial counts/volume of cream was the
same. Controls were performed with cream without the addition of
bacterial cultures or with cream with the addition of 40 mL of calcium-
alginate beads without bacteria. Fermentation was performed at 30 ◦ C
for 15 h in an incubator (TH 30, Edmund Buehler GmbH) in combination
with a universal shaker (80 RPM, Edmund Buehler GmbH). The calcium-
alginate beads were separated from the cream by centrifugation
(4000×g, 5 min) and washed twice with sterile distilled water.
The strain L. paracasei L2A21K5 showed the greatest reduction of
cholesterol in cream and was therefore selected for butter production.
Fermentation was performed according to the procedure described
above using 400 mL of calcium-alginate beads containing the immobi­
lized strain L2A21K5 and 800 mL of pasteurized cream. After fermen­
tation, the cream was separated from the alginate beads using a sieve
under vacuum. The cream was whipped with a mechanical butter churn
(Manvelb A20; Elecrem, Navnes, France) until butter granules were
visible. The buttermilk was drained and the butter was washed with
distilled water. Salt (2%) was then added, and the butter was shaped and
stored at 4 ◦ C. Butter making was performed in triplicate on different
days with different batches of fermented cream. The fermented cream
and the butter produced from it were analyzed for bacterial counts, pH,
cholesterol content and fatty acid composition.
2.5. Bacterial counts
Total number of viable bacteria in cream and calcium-alginate beads
were enumerated in duplicate on MRS agar (Biokar Diagnosis, France).
Aliquots of cream (1 mL) or calcium-alginate beads (1 g) from each
treatment were placed in tubes containing 9 mL of sterile peptone water
(Biokar Diagnosis, France). The suspension of calcium-alginate beads
was shaken until the beads were completely dissolved to release the
bacteria. Serial decimal dilutions of suspensions were made with sterile
peptone water, plated in duplicate in MRS agar (Biokar Diagnosis,
France) and incubated at 30 ◦ C for 48 h. Data were expressed as colony
forming units per mL (CFU/mL). Analysis were performed in duplicate
in three independent experiments.
2.6. Cholesterol and fatty acid analysis
Fat was extracted from cream and butter by the Folch method (Folch,
Lees, & Sloane Stanley, 1957) with minor modifications. Briefly, ali­
quots (2.5 mL of cream or 1 g of butter) were transferred into 50 mL
volumetric flasks with 32 mL of a mixture of chloroform/methanol (1:1,
v/v). Each flask was placed in a boiling water bath until the contents of
the flask began to boil. The flasks were then removed, allowed to cool to
room temperature before making up to volume (50 mL) with chloro­
form. The contents of each flask were then filtered (Paper filter What­
man 1), mixed thoroughly with 15 mL of water and the mixture was
allowed to separate into two phases. The organic phase (lipids in chlo­
roform) was collected and extracts were dried (Rotary Evaporator Buchi,
model R-251) under low pressure (200 mBar) at 37 ◦ C to remove the
solvent. The lipid extracts were then diluted with chloroform (1 mL).
The chloroform extract containing the cholesterol was then determined
by the Liebermann-Burchard colorimetric method (Kenny, 1952). A
calibration curve was performed with cholesterol standards (0.5–4 mg/
mL, R2 = 0.99992).
Fatty acid analysis was carried out by gas chromatography (with a
M.F.B. Teixeira et al.
fused silica capillary column and a flame ionization detector) according
to Silva et al. (2019). Briefly, FA methyl esters were prepared by
transesterification with sodium methoxide. Fatty acid methyl esters
were analyzed using a Shimadzu 2010 Plus gas chromatograph (Shi­
madzu, Kyoto, Japan), equipped with a flame ionization detector and a
fused silica capillary column SP-2560 (100 m, 0.2 mm, 0.20 μm, Supelco
Inc., Bellefonte, PA, USA). Helium was used as carrier gas and the split
ratio was 1:50. Injector and detector temperatures were set at 250 ◦ C
and 280 ◦ C, respectively. The initial oven temperature of 50 ◦ C was held
for 1 min, then increased at 50 ◦ C/min to 150 ◦ C, where it was held for
20 min, then increased at 1 ◦ C/min to 220 ◦ C and held for 30 min. Ex­
tractions and analyses were performed in duplicate for each sample.
2.7. Sensory analysis
Sensory analysis was performed on two types of butter: Butter pro­
duced after fermentation of the cream (with reduced cholesterol con­
tent) and a commercial Azorean butter (purchased in the local market).
This evaluation was performed by 28 semi-trained tasters in accordance
with Annex IV (Article 4) of Commission Regulation (EC) No. 273/2008
(Commission Regulation, 2008) The tasters were butter consumers
found to be competent in sensory classification. First, the methods,
scales, and description of sensory impressions were explained according
to the reference method described in Annex IV of the Commission
Regulation. Butter samples were stored and coded at room temperature
(20 ◦ C) for 10 min prior to testing so that the identity of the samples was
not disclosed during evaluation. Sensory testing focused on the
following three attributes: Appearance, consistency, and flavor.
Appearance included the following attributes: Color, visible purity,
absence of physical impurities, absence of mold, and uniformity of water
dispersion. Consistency includes the following characteristics: Fullness,
texture and firmness. The spreadability of the butter was tested by
physical means (knife and spreadable toast). The scale used consisted of
a score from 1 to 5, with 5 representing “very good” (ideal type, superior
quality), 4 representing “good” (no obvious defects), 3 representing
“fair” (slight defects), 2 representing “weak” (obvious defects), and 1
representing “very weak” (obvious defects).
2.8. Statistical analysis
To assess the effect of treatments (alginate beads), data of pH,
cholesterol reduction, bacterial counts and fatty acid composition were
analyzed by one-way analysis of variance (ANOVA). When statistically
significant differences (p < 0.05) were found in the ANOVA, the Tukey
test was applied for multiple comparisons between strains. In the sen­
sory evaluation, the non-parametric Wilcoxon-Mann-Whitney test for
independent samples was used. All statistical analysis was performed
using the SPSS program (IBM SPSS Statistics, Version 24).
3. Results & discussion
3.1. Application of bacterial cultures in cream fermentation
In the present study, two fermentation methods were compared: the
application of bacterial cultures directly in the cream and the incorpo­
ration of bacteria into the calcium-alginate beads to ferment the cream.
The results of these two methods are shown in Fig. 1. Fermentation of
cream by L. paracasei strains incorporated into the calcium-alginate
beads reduced cholesterol content by 17–31%. In contrast, the reduc­
tion in cholesterol content was negligible when free bacterial cells were
applied to cream fermentation. For example, application of the
L. paracasei L2A21K5 strain to cream fermentation showed a 4.3 ± 2.7 %
reduction in cholesterol. However, when this strain was incorporated
into the calcium-alginate beads, there was a cholesterol reduction of
31.1 ± 4.8 %. A control with calcium-alginate beads without the addi­
tion of LAB was also performed, but no changes in the cholesterol
3
Food Chemistry 393 (2022) 133419
Fig. 1. Cholesterol reduction (%) in cream fermented by L. paracasei strains
L2A21K5, L3B1M2 and L3B21R1, immobilized in calcium-alginate beads
(immobilized cells) or applied directly to cream (free cells). The values shown
are the average of three assays (analyzed in duplicate) and the error bars
correspond to the standard error of the mean (SEM). Different lowercase letters
indicate significantly differences (p < 0.05) between strains and different up­
percase letters indicate significantly differences (p < 0.05) between treatments.
content of the cream were observed (results not shown).
Some studies have reported that the reduction of cholesterol by LAB
is due to the adhesion of cholesterol to the cell wall (Bosch et al., 2014)
and/or its incorporation into the cell membrane (Anandharaj & Siva­
sankari, 2014). In the present work, when LAB was added to cream and
not removed after fermentation, no significant reduction in the amount
of cholesterol in the cream was observed. This observation confirms that
the cholesterol was not metabolized but remained associated with the
bacterial cells. However, the use of LAB, immobilized in calcium-
alginate beads, allowed not only the uptake and incorporation of
cholesterol by bacteria, but also the separation of the bacterial cells at
the end of the process. Therefore, this process proved to be effective in
removing cholesterol from cream during fermentation. Few authors
have used bacterial strains in the fermentation of cream to reduce
cholesterol. Aloğlu and Öner (2006) reported cholesterol assimilation by
LAB in cream, which ranged from 20.62 to 59.78% after 24 h of
fermentation. In his study, the cream, which contained 15% fat, was
centrifuged at high speed (12000 × g) at the end of the incubation period
to remove the bacterial cells. However, for the production of butter, a
higher percentage of fat must be used in the cream, which limits the
removal of bacterial cells by centrifugation. In their study, the amount of
cholesterol assimilated in cream (35% fat content) used for butter pro­
duction was significantly reduced and ranged from 0.14% to 25.35%
when the pH was lowered from 4.60 to 3.96 (Aloğlu and Öner, 2006).
The decrease in pH (acidification of the cream) and the leakage of
bacterial cells from the calcium-alginate beads into the fermented cream
were monitored and shown in Fig. 2. The pH values after fermentation
ranged from 4.44 to 4.63, while the unfermented cream had a higher pH
of 5.58. No differences (p > 0.05) were observed in pH values between
the two processes (with and without alginate beads). These results
indicate that substrates and products such as lactose and lactic acid
diffuse through the alginate gel to enable the fermentation process.
The use of calcium alginate entrapped LAB has been described by
several authors in various applications such as milk fermentation for
yogurt production or cream fermentation for the production of fer­
mented butter (Champagne & Côté, 1987; Prevost, Divies, & Rousseau,
1985; Stenroos, Linko, & Linko, 1982). As reported by these authors,
calcium alginate is non-toxic and allows the immobilized LAB to grow
and maintain normal metabolism such as the production of lactic acid.
The pH values observed were slightly lower than those reported by
Champagne and Côté (1987), indicating a high acidifying capacity of the
lactobacilli studied. However, Aloğlu and Öner (2006) reported that
LAB cultures have a higher ability to remove cholesterol when the pH
decreases to more acidic values (pH < 4). However, sour cream has a
M.F.B. Teixeira et al. Food Chemistry 393 (2022) 133419

Fig. 2. A) Values of pH in cream fermented by L. paracasei strains L2A21K5, L3B1M2 and L3B21R1 immobilized in calcium-alginate beads (immobilized cells) or
applied directly to cream (free cells). B) Bacterial counts (Log CFU/g) in calcium-alginate beads and cream during fermentation. Results represent the mean ± SEM
calculated from three independent experiments. Different lowercase letters indicate significantly differences (p < 0.05) between strains and different uppercase
letters indicate significantly differences (p < 0.05) between treatments.

negative effect on the taste of cream and butter (Champagne and Côté,
1987).
The number of bacteria in the calcium-alginate beads and the
leakage into the cream during fermentation are shown in Fig. 2B. Bac­
terial counts in the calcium-alginate beads were significantly (p > 0.05)
higher after fermentation of cream, by about 1 log unit. This is consistent
with the study of Champagne and Côté (1987), which reported that
calcium-alginate beads allow bacterial growth up to a level of 109/g, at
which it stabilizes. Pereira and Gibson (2002) indicated that the ability
of bacteria to assimilate cholesterol is highly dependent on their growth.
Thus, the ability of calcium-alginate beads to allow the growth of LAB
could be beneficial for cholesterol uptake by cells. Only a small fraction
(0.41%) of the original bacterial population was released into the cream
at the end of fermentation. These results are consistent with other
studies in which bacterial cells were entrapped in alginate beads. Ac­
cording to Champagne and Côté (1987), only a small fraction of the
immobilized LAB (0.4% of the initial population) was released from the
calcium-alginate beads into the cream after fermentation. In a similar
study by Prevost and Divies (1992), cell leakage from the gel into the
fermented cream accounted for only 0.15% of the total cells entrapped.
Therefore, most of the cholesterol taken up by LAB remained entrapped
and can be removed from the cream after separation of the alginate
beads. In addition, cream fermented with immobilized LAB would be
less susceptible to acidification during storage due to its lower bacterial
content (Champagne and Côté, 1987).
3.2. Production of low-cholesterol butter
To produce butter with reduced cholesterol content, the strain
L. paracasei L2A21K5 was selected and included in alginate for cream
fermentation. The results of cholesterol content in butter produced from
sour and sweet cream (cultured butter and sweet cream butter) are
shown in Fig. 3. The butter produced with fermented cream had 44%
lower cholesterol content (68 mg cholesterol per 100 g butter) compared
to the control (sweet cream butter – 121 mg cholesterol per 100 g but­
ter). Several authors reported large differences in the cholesterol content
of butter from different origins, ranging from 148 to 369 mg/100 g.
(Capuano, Gravink, Boerrigter-Eenling, & van Ruth, 2015; Derewiaka,
Sosińska, Obiedziński, Krogulec, & Czaplicki, 2011; Gonçalves & Bag­
gio, 2012; Kolarič & Šimko, 2020; Pustjens, Boerrigter-Eenling, Koot,
Rozijn, & Van Ruth, 2017; Seçkin, Gursoy, Kinik, & Akbulut, 2005). The
butter cholesterol levels obtained in this study were lower than those
reported by these authors, but previous studies have also shown that
Azorean butter based on grass pasture systems tends to have lower
cholesterol content (Silva et al., 2019).
Few authors have investigated the use of cholesterol-assimilating
LAB in the production of cholesterol-reduced butter. Aloğlu and Öner
(2006) used the probiotic LAB to assimilate cholesterol in cream, but the
cholesterol content of the resulting butter varied between 187 and 205
mg/100 g. Recently, Kim et al. (2021) used a strain of Pediococcus
Fig. 3. Cholesterol content in butter produced from sweet cream (control
butter) and sour cream obtained by fermentation with L. paracasei L2A21K5
immobilized in alginate beads (cultured butter). The values shown are the
average of three assays (analyzed in duplicate) and the error bars correspond to
the standard error of the mean (SEM). Different letters indicate significantly
differences (p < 0.05).
acidilactici in melted butter but achieved only a small reduction in
cholesterol (4.2%). The addition of bile salts to the butter increased the
cholesterol reduction to 11% of the initial concentration, but this could
have implications for the sensory quality of the butter.
To evaluate the possible effects of the bacteria entrapped in the
calcium-alginate beads on the quality of the butter, the fatty acid (FA)
profile of the resulting butter was compared with that of the cream used
before fermentation (Table 1). In general, the concentrations of each FA
showed no significant differences (p > 0.05) between sour cream butter
and sweet cream butter, with a few exceptions. Beneficial FAs such as
butyric (C4:0), stearic (C18:0), oleic (18:1), monounsaturated (MUFA)
and n-3 FAs (C18:3n-3 and C20:5n-3) showed a slight increase (p < 0.05)
in cultured butter (Table 1). In contrast, cultured butter exhibited a
slight but significant (p < 0.05) reduction in n-6/n-3 ratio and saturated
FA (SFA), including myristic and palmitic acids (C14:0 and C16:0,
respectively). In addition, both atherogenic index (AI) and thromboge­
nicity index (TI) were significantly reduced (p < 0.05) in cultured butter
(Table 1). The AI denotes the ratio between saturated FAs, which are
considered pro-atherogenic, and unsaturated FAs with anti-atherogenic
properties, while TI is defined by the ratio between pro-thrombogenic
FA (SFA) and anti-thrombogenic FAs and indicates the tendency to
form clots in blood vessels (Ghaeni, Ghahfarokhi, & Zaheri, 2013).
Overall, these results suggest that incorporation of cholesterol into the
membranes of growing bacteria during cream fermentation reduces the
uptake of unsaturated FA, especially n-3 FA, and increases the uptake of
SFAs, resulting in a butter with a healthier composition (lower SFA, AI,
and TI). Current dietary guidelines recommend reducing SFA intake to
avoid an increase in serum LDL cholesterol concentration, which is

4
M.F.B. Teixeira et al. Food Chemistry 393 (2022) 133419

Table 1 Table 2
Fatty acid composition of cream and butter (g/100 g fat). Sensory analysis of low cholesterol and commercial butters. Values are the
Fatty acid Cream Butter p-value1
average of scores given on a 1–5 scale.
Sensory attribute Low cholesterol butter Commercial butter p-value
C4:0 1.46 ± 0.04 1.60 ± 0.01 0.038
C6:0 1.50 ± 0.01 1.53 ± 0.00 0.091 Appearance 3.68 4.63 <0.001
C8:0 1.08 ± 0.03 1.07 ± 0.00 0.711 Consistency 3.96 4.70 <0.001
C10:0 2.69 ± 0.12 2.54 ± 0.00 0.254 Flavor/aroma 3.96 4.52 0.006
C11:0 0.29 ± 0.02 0.26 ± 0.00 0.378
C12:0 3.16 ± 0.18 3.45 ± 0.00 0.171
C13:0 0.07 ± 0.01 0.07 ± 0.00 0.795 appearance, visible water droplets, not homogeneous, too intense
C14:0 11.61 ± 0.17 11.06 ± 0.02 0.030
coloration, grainy, marbled, and undissolved salt. These defects were
C15:0 0.91 ± 0.08 0.97 ± 0.01 0.520
C16:0 29.51 ± 0.91 25.95 ± 0.08 0.018 probably due to the artisanal production method of this butter, espe­
C17:0 0.48 ± 0.01 0.51 ± 0.00 0.135 cially in the kneading phase. In general, however, the tasters found this
C18:0 12.14 ± 0.39 13.80 ± 0.04 0.014 butter to have good consistency and flavor (average score 3.96).
C20:0 0.13 ± 0.01 0.15 ± 0.00 0.158
C21:0 0.04 ± 0.01 0.04 ± 0.00 1.000
C22:0 0.05 ± 0.00 0.07 ± 0.00 0.007 4. Conclusion
C23:0 0.02 ± 0.00 0.03 ± 0.01 0.678
C24:0 0.04 ± 0.00 0.04 ± 0.00 0.230 The present study shows that incorporation of cholesterol-
C12:1 0.07 ± 0.01 0.07 ± 0.00 0.519
C14:1c9 0.84 ± 0.05 0.75 ± 0.01 0.114
assimilating LAB into calcium-alginate beads for fermentation of
C16:1c7 0.19 ± 0.00 0.22 ± 0.00 0.003 cream could be an effective and innovative method to remove choles­
C16:1c9 1.13 ± 0.02 1.05 ± 0.00 0.014 terol from the final product. The high fat content of cream makes it
C17:1c9 0.19 ± 0.00 0.20 ± 0.00 0.016 difficult to remove the bacterial cells by centrifugation, but by using
C18:1c9 21.42 ± 0.42 22.99 ± 0.06 0.021
calcium-alginate beads, the bacterial cells can be easily separated from
C18:1c11 0.47 ± 0.03 0.48 ± 0.00 0.839
C18:1c12 0.12 ± 0.00 0.11 ± 0.00 0.101 the cream. Thus, it was possible to effectively reduce the cholesterol
C18:1c13 0.05 ± 0.00 0.05 ± 0.00 0.374 content in the fermented cream. The final product was a low-cholesterol
C18:1c15 0.08 ± 0.02 0.08 ± 0.00 0.842 butter (reduction of 44%) obtained from cream fermented with
C20:1 0.08 ± 0.00 0.09 ± 0.00 1.000 L. paracasei L2A21K5 immobilized in calcium-alginate beads. In addi­
C16:1t9 0.12 ± 0.01 0.13 ± 0.00 0.374
C18:1t6/t7/t8 0.25 ± 0.01 0.27 ± 0.00 0.124
tion to the reduction in cholesterol content, this butter had improved FA
C18:1t9 0.27 ± 0.07 0.22 ± 0.00 0.459 composition, including better atherogenic and thrombogenicity indices,
C18:1t10 0.34 ± 0.02 0.36 ± 0.01 0.488 and was rated good in sensory tests of appearance, consistency, and
C18:1t11 2.57 ± 0.36 2.68 ± 0.00 0.767 flavor.
C18:1t12 0.33 ± 0.03 0.39 ± 0.01 0.072
C18:1t15 0.26 ± 0.01 0.27 ± 0.00 0.795
C18:1t16 0.29 ± 0.01 0.33 ± 0.00 0.024
C18:2n-6 1.43 ± 0.08 1.42 ± 0.02 0.971 Declaration of Competing Interest
C18:3n-3 0.51 ± 0.01 0.57 ± 0.01 0.029
C20:2n-6 0.03 ± 0.00 0.03 ± 0.00 1.000
The authors declare the following financial interests/personal re­
C20:3n-6 0.06 ± 0.00 0.07 ± 0.00 0.016
C20:4n-6 0.09 ± 0.00 0.10 ± 0.01 0.275 lationships which may be considered as potential competing interests:
C20:5n-3 0.04 ± 0.00 0.05 ± 0.00 0.016 Celia C. G. Silva reports financial support was provided by Portuguese
CLA 1.08 ± 0.17 1.06 ± 0.00 0.898 Foundation for Science and Technology. Sofia P. M. Silva reports
SFA 65.20 ± 0.54 63.12 ± 0.08 0.019 financial support was provided by Portuguese Foundation for Science
MUFA (cis) 24.63 ± 0.38 26.08 ± 0.06 0.019
MUFA (trans)2 4.42 ± 0.42 4.65 ± 0.02 0.618
and Technology. Jose A. M. Prates reports financial support was pro­
PUFA 2.15 ± 0.10 2.25 ± 0.04 0.399 vided by Portuguese Foundation for Science and Technology. Marina F.
n-3 FA3 0.54 ± 0.01 0.62 ± 0.01 0.010 P. Domingos-Lopes reports financial support was provided by Fundo
n-6 FA4 1.60 ± 0.09 1.62 ± 0.0 0.841 Regional da Ciência e Tecnologia.
n-6/n-3 2.95 ± 0.10 2.63 ± 0.03 0.042
Atherogenic index (AI)5 2.54 ± 0.06 2.23 ± 0.01 0.006
Thrombogenicity index (TI)6 2.68 ± 0.06 2.46 ± 0.02 0.026 Data availability
1
A p value < 0.05 indicate statistical differences.
2 Data will be made available on request.
Sum of t9-C16:1, t6/7/8-, t9-, t10-, t11-, t12-, t15-and t16-C18:1.
3
Sum of C18:3n-3 and C20:5n-3.
4
Sum of C18:2n-6, C20:2n-6, C20:3n-6 and C20:4n-6. Acknowledgments
5
AI = (C12:0 + 4 × C14:0 + C16:0)/(MUFA + PUFA).
6
TI = (C14:0 + C16:0 + C18:0)/(0.5 × MUFA + 0.5 × n-6 + 3 × n-3 + n-6/n- This work was financed by Portuguese Foundation for Science and
3). Technology – FCT, Project UID/CVT/0153/2016 and UIDB/00276/
2020 (CIISA). S.P.M. Silva received support through grant SFRH/BD/
associated with an increased risk of coronary heart disease (Griffin, 139525/2018 from the Portuguese Foundation for Science and Tech­
Mensink, & Lovegrove, 2021; Yanai & Tada, 2018). nology (FCT). M.F.P. Domingos-Lopes received support through grant
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