Bioprocess of astaxanthin extraction from shrimp waste via the common microorganisms Saccharomyces cerevisiae and Lactobacillus acidophilus in comparison to the chemical method

Abstract

This study compared microbiological and chemical methods used in astaxanthin extraction from the exoskeleton of the shrimp species Penaeus japonicus and Penaeus semisulcatus. The microbiological method was performed using Saccharomyces cerevisiae (bakery yeast) or Lactobacillus acidophilus (from yogurt), followed by solvent extraction with hexane and acetone at different ratios (1:1, 1:2, and 1:3). The chemical method was performed traditionally using hexane. The highest astaxanthin yield from P. japonicus exoskeleton was obtained using either S. cerevisiae or L. acidophilus followed by solvent extraction with hexane and acetone at a ratio of 1:1 (8.5 and 8.1 mg/g waste, respectively) as well as by the chemical method (8.4 mg/g waste). Likewise, the highest astaxanthin yield from P. semisulcatus exoskeleton was obtained using either S. cerevisiae or L. acidophilus followed by solvent extraction with hexane and acetone at a ratio of 1:1 (3.0 and 4.1 mg/g waste, respectively) as well as by the chemical method (3.2 mg/g waste). The values obtained from P. semisulcatus exoskeleton were considerably lower than those attained from P. japonicus exoskeleton. In addition, the nuclear magnetic resonance (C-NMR) analysis confirmed that astaxanthin was the main carotenoid present in the extract. In conclusion, the pretreatment of exoskeleton wastes of P. japonicus using S. cerevisiae followed by solvent extraction with hexane and acetone at a ratio of 1:1 as well as the classical chemical treatment led to the highest astaxanthin content.

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Biomass Conversion and Biorefinery
https://doi.org/10.1007/s13399-022-02984-2
ORIGINAL ARTICLE
Bioprocess ofastaxanthin extraction fromshrimp waste
viathecommon microorganisms Saccharomyces cerevisiae
andLactobacillus acidophilus incomparison tothechemical method
SalwaA.H.Hamdi1· GhadeerM.Ghonaim2· RanaR.ElSayed2· SusanaRodríguez‑Couto3·
MohamedN.AbdEl‑Ghany4
Received: 22 March 2022 / Revised: 20 June 2022 / Accepted: 21 June 2022
© The Author(s) 2022
Abstract
This study compared microbiological and chemical methods used in astaxanthin extraction from the exoskeleton of the shrimp
species Penaeus japonicus and Penaeus semisulcatus. The microbiological method was performed using Saccharomyces
cerevisiae (bakery yeast) or Lactobacillus acidophilus (from yogurt), followed by solvent extraction with hexane and acetone
at different ratios (1:1, 1:2, and 1:3). The chemical method was performed traditionally using hexane. The highest astaxanthin
yield from P. japonicus exoskeleton was obtained using either S. cerevisiae or L. acidophilus followed by solvent extraction
with hexane and acetone at a ratio of 1:1 (8.5 and 8.1mg/g waste, respectively) as well as by the chemical method (8.4mg/g
waste). Likewise, the highest astaxanthin yield from P. semisulcatus exoskeleton was obtained using either S. cerevisiae or L.
acidophilus followed by solvent extraction with hexane and acetone at a ratio of 1:1 (3.0 and 4.1mg/g waste, respectively) as
well as by the chemical method (3.2mg/g waste). The values obtained from P. semisulcatus exoskeleton were considerably
lower than those attained from P. japonicus exoskeleton. In addition, the nuclear magnetic resonance (C-NMR) analysis
confirmed that astaxanthin was the main carotenoid present in the extract. In conclusion, the pretreatment of exoskeleton
wastes of P. japonicus using S. cerevisiae followed by solvent extraction with hexane and acetone at a ratio of 1:1 as well as
the classical chemical treatment led to the highest astaxanthin content.
Keywords Astaxanthin· Shrimp waste· Carotenoid, Saccharomyces cerevisiae· Lactobacillus acidophilus
1 Introduction
Crustaceans are one of the oldest and the most diverse group
of arthropods. They are also considered one of the most suc-
cessful groups of invertebrates on Earth [1]. Crustaceans
are regarded as an enriched source of many bioactive sub-
stances, such as carbohydrates, proteins, amino acids, fatty
acids, vitamins, and minerals, especially the Decapoda order
within the Malacostraca class. This order includes many
familiar groups such as shrimps, prawns, crabs, lobsters,
and crayfish, which have well-known nutritional function
and importance. They are also delicious and easily digest-
ible. In addition, their nutritional function depends on the
biochemical composition of their bodies, which consists of
high protein, low fat, and carbohydrate contents similar to
those in fish flesh. Moreover, crustaceans are considered a
source of omega-3 fatty acids, and vitamins, including A, B,
especially B3 (niacin) and B12 (cobalamin). Hence, they are
among the most valuable components of the human diet [2].
* Susana Rodríguez-Couto
Susana.Rodriguez.Couto@lut.fi
* Mohamed N. Abd El-Ghany
mabdelghany@sci.cu.edu.eg
1 Zoology Department, Faculty ofScience, Cairo University,
Giza12613, Egypt
2 Department ofBiotechnology andBimolecular Chemistry,
Cairo University, Giza12613, Egypt
3 Department ofSeparation Science, LUT School
ofEngineering Science, LUT University, 50130Mikkeli,
Finland
4 Botany andMicrobiology Department, Faculty ofScience,
Cairo University, Giza12613, Egypt
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Biomass Conversion and Biorefinery
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One of the major problems in modern food production is
the generation of large quantities of underused by-products.
These food wastes may contain substances of high value
and important health benefits. Thus, the seafood processing
industry, for example, produces a tremendous quantity of by-
products and wastes, such as heads, tails, skins, scales, vis-
cera, backbones, and shells that may be an amazing source
of proteins, lipids, and pigments [3]. In addition, shells may
be a source of chitinous materials and carotenoids. There-
fore, the waste generated during food processing should be
utilized as it is considered a wasted fortune [4]. This would
also alleviate the problems generated by their disposal [5].
Carotenoids such as astaxanthin, beta-carotene, lutein,
and others are extracted from crustacean exoskeletons. They
are responsible for the pigmentation of most aquatic organ-
isms. Some carotenoids are precursors of vitamin A. They
can also act as antioxidants in the biological systems [6],
and exhibit protective action against cancer [7]. In the cara-
pace of crustaceans, carotenoids exist as free and esterified
forms. Many crustaceans can produce astaxanthin from
beta-carotene ingested from dietary algae via echinenone
3-hydroxyechinenone, canthaxanthin, and adonirubin, as
shown in Fig.1 [8]. Thus, astaxanthin has been reported to
be 10 times larger than that of any other carotenoids such as
zeaxanthin, lutein, and canthaxanthin [9].
Currently, astaxanthin is a renowned compound for its
commercial application in various industries, comprising
aquaculture, food, cosmetics, nutraceutical, and pharma-
ceutical [10]. Moreover, astaxanthin effectively suppresses
cell damage caused by free radicals, and induction of matrix
metalloproteinases (MMPs) in skin after UV irradiation
[11]. It repairs DNA damage caused by skin exposure to
UV radiation, which can lead to oncogenic mutations. Stud-
ies showed that astaxanthin inhibited the UV-induced DNA
damage and increased the expression of oxidative stress-
responsive enzymes [12]. Astaxanthin is also reported to
be an inhibitor of matrix metalloproteinases (MMPs) in dif-
ferent cells, including macrophages and chondrocytes [13].
Fig. 1 Metabolism of β-carotene in crustaceans
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Biomass Conversion and Biorefinery
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It has anti-inflammatory properties as it inhibits the gene
expression of several proinflammatory biomarkers, such
as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor
necrosis factor-α (TNF-α) in Alzheimer disease (AD), ani-
mal model [14]. Furthermore, astaxanthin has anti-aging
effects such as hyper-pigmentation suppression, melanin
synthesis, photoaging inhibition, and wrinkle formation
reduction [15]. Astaxanthin has antioxidant activity since it
inhibits reactive oxygen species (ROS) formation and modu-
lates the expression of oxidative stress-responsive enzymes
such as heme oxygenase-1 (HO-1) [12, 16]. Astaxanthin has
immune-enhancing effects as it increases natural killer (NK)
cell cytotoxic activity [17], suggesting that it may regulate
NK cells that serve as an immunosurveillance system against
tumors and virus-infected cells [18]. In addition, astaxanthin
supplementation was proven to be useful to heart failure
patients with left ventricular systolic dysfunction (LVSD).
It was found that after 3months of astaxanthin supplemen-
tation in patients with LVSD, the levels of the oxidative
stress markers decreased, and both cardiac contractility and
exercise tolerance improved [19]. The recommended dose
of astaxanthin for adult patients is 2–4mg/day. In addition,
astaxanthin is safe and has no side effects when consumed
with food [20].
Khanafari etal. [20] used the Lactobacillus species Lac-
tobacillus plantarum and Lactobacillus acidophilus for the
extraction of astaxanthin from wastes of the shrimp species
Penaeus semisulcatus. Also, Hamdi etal. [21] used Lacto-
bacillus and Saccharomyces for the extraction of astaxan-
thin from byproducts of the crayfish Procambarus clarkia.
Consequently, in the present research, Lactobacillus and
Saccharomyces species were assessed as pretreatment for
the extraction of astaxanthin from shrimp wastes as an eco-
friendly alternative to the classical chemical method. The
shrimp species selected, namely, Penaeus japonicus and
Penaeus semisulcatus, are two commercially used shrimps
in Egypt.
2 Materials andmethods
2.1 Sample preparation
Fresh shrimps of two different species, Penaeus japonicus
and Penaeus semisulcatus, were purchased from a local
fish market. The shrimps were peeled, and all the internal
organs were removed in order to obtain the exoskeleton
(waste). Shrimp waste was then dried in an oven for 8–10h
at 55–60°C [22]. The dried waste was ground to obtain a
fine powder (1–3mm particle size). The process was per-
formed separately for each species. The powder samples
were collected in sterilized containers, labeled, and stored
in a fridge at 4°C.
2.2 Microorganisms andculture media
Lactobacillus acidophilus was obtained from a commercial
yogurt and identified biochemically according to Pyar and
Peh [23]. Saccharomyces cerevisiae was obtained from a
commercial bakery yeast in Egypt and identified biochemi-
cally and microbiologically. Czapek-Dox medium con-
sisted of sucrose 20.0g/L, sodium nitrate 2.0g/L, dipo-
tassium phosphate 1.0g/L, magnesium sulfate 0.50g/L,
potassium chloride 0.50g/L, ferrous sulfate 0.01g/L, and
agar 15.0g/L. De Man, Rogosa, and Sharpe (MRS) broth
medium was composed of peptone from casein 10.0g/L,
yeast extract 4.0g/L, meat extract 8.0g/L, D ( +) glucose
20.0g/L, Tween 80 1.0g/L, di-ammonium hydrogen citrate
2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g/L,
manganese sulfate 0.04g/L, and agar 15g/L. The media
were sterilized by autoclaving at 121°C and 1.5 bars for
15min. Streptomycin (30mg/mL) that was previously steri-
lized by filtration (0.2µm) was added after cooling down in
order to prevent bacterial contamination. All the used flasks
were cleaned by soaking them overnight in diluted H2SO4,
followed by ethanol, and distilled water washing. Flasks
(250mL) containing 100mL of Czapek-Dox medium were
inoculated with 5g of instant dry yeast and incubated for
5days at 35°C [24]. Flasks (250mL) containing 100mL of
MRS medium were inoculated with 1mL of L. acidophilus
and incubated for 3days at 30°C [22].
2.3 Shrimp waste bioprocess
5mL of previously cultured MRS broth containing L. aci-
dophilus, and 5mL of Czapek-Dox medium containing S.
cerevisiae were added to a fermentative medium (100mL
distilled water + 10g of the ground exoskeletons) and incu-
bated for 5days at 30°C in the presence of 5% CO2 [25].
The fermentative medium was filtered with Whatman filter
paper no. 41 and centrifuged at 3075g for 5min. 25mL of
the filtered fermentation medium was added to 25mL of a
mixture of hexane and acetone at different ratios (1:1, 1:2,
and 1:3) to determine the one leading to the highest astax-
anthin yield. The entire extraction process was performed
separately for each species of shrimp. The obtained extracts
were used for astaxanthin determination.
2.4 Chemical extraction
Astaxanthin was extracted by mixing 10g of the ground
exoskeletons of each shrimp species with 50mL of hexane
in a 100-mL flask, vortexed for 30s, and placed in a 50°C
water bath for 10min. Aqueous and organic layers were
separated by centrifugation at 1008g for 5min. In the final
step, 6mL of dimethyl sulfoxide (DMSO) were added to the
tube, vortexed vigorously, placed in a water bath for 10min,
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Biomass Conversion and Biorefinery
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and then vortexed again. The obtained extracts from each
shrimp species were used for astaxanthin determination.
2.5 Determination ofastaxanthin concentration
The astaxanthin contained in the extracts from each shrimp
species obtained via all microbial and chemical methods was
determined by UV–VIS spectrometry (Jenway 6300 spectro-
photometer) at a wavelength of 476nm [26]. In the blank,
the extract was replaced by distilled water [27].
2.6 HPLC analysis
The astaxanthin content of the extracts was determined by
high-performance liquid chromatography (HPLC) (Agilent
1260 series) equipped with a diode array detector (DAD) and
an Eclipse C18 column (250mm × 4.6mm, 5µm, Eclipse).
The mobile phase consisted in water (A) and 0.05% of tri-
fluoroacetic acid in acetonitrile (B), and the samples were
eluted at a flow rate of 1mL/min for 8min with an isocratic
gradient. The injection volume was 5 μL for both samples
and standard. The column temperature was maintained at
40°C. The UV detection of the elute was performed at
480nm. Astaxanthin was qualitatively analyzed by com-
paring the retention time of the standard, and its quantifica-
tion was done by using a calibration curve. This work was
conducted at the National Research Centre, Cairo, Egypt.
2.7 C‑NMR studies
The extracts from shrimp exoskeletons via the microbial and
chemical methods were subjected to C-NMR analysis. The
results were compared to the standard astaxanthin C-NMR
analysis [28]. Dimethyl sulfoxide (DMSO) was used as a sol-
vent. All NMR spectra were recorded on a Bruker Advance
III 400MHz. Chemical shifts were reported relative to TMS
and referenced via residual carbon resonances of the appro-
priate deuterated solvent.
2.8 Statistical analysis
The matrix of the factorial experimental design is presented
in Supplementary Table2. The statistical analysis and the
ANOVA of the experimental design were performed using
the SPSS statistics software version 25 (Supplementary
Tables3 and 4). All determinations were carried out in trip-
licate. All data were expressed as mean ± SD. at (p < 0.001).
The standard deviation of the samples was calculated using
IBM SPSS statistics software version 25.
3 Results
3.1 Astaxanthin concentration intheextracts
3.1.1 UV–VIS spectroscopy
Astaxanthin concentrations in the extracts of P. japonicus
exoskeleton were higher than 8.0mg/g waste using either
S. cerevisiae or L. acidophilus followed by solvent extrac-
tion with hexane and acetone at a ratio of 1:1 as well as by
the chemical method (Fig.2A). However, astaxanthin from
the extracts of P. semisulcatus exoskeleton led to much
lower values (3.0mg/g waste with S. cerevisiae followed
by solvent extraction with hexane and acetone at a ratio
of 1:1, 4.1mg/g waste with L. acidophilus followed by
solvent extraction with hexane and acetone at a ratio of 1:1
and 3.2mg/g waste with the chemical method) (Fig.2B).
It is noteworthy that the microbiological method followed
by solvent extraction with hexane and acetone at a ratio of
1:2, and especially at 1:3, acutely decreased the extracted
astaxanthin (Fig.2).
3.1.2 HPLC
HPLC chromatographs of the extracts are presented in
Supplementary Fig.3. The obtained results (Table1)
were much lower than those determined by UV–VIS spec-
troscopy shown in Fig.2. The reason for this is that the
UV–Vis method does not enable the distinction between
carotenoids in a given mixture and the most common
protocols use a mean absorption coefficient and a mean
absorption wavelength. Therefore, more accurate results
are attained by the HPLC method for specific carotenoids.
3.1.3 C‑NMR studies
The C-NMR analysis (Supplementary Fig.4) of astax-
anthin extracted from P. japonicus and P. semisulcatus
by chemical and microbiological methods confirmed that
astaxanthin was the main carotenoid present in the sam-
ples. The two peaks, between 7.004 and 8.557ppm, sym-
bolized the presence of protons of methine on the astaxan-
thin main chain [29]. These peaks proved the presence of
astaxanthin as two sets in the monoesterified compounds
due to the loss of symmetry. At 2.505ppm, four protons
corresponded to the methylene protons, and α to the car-
bonyl. Signals at 2.022 and 1.83ppm corresponded to
the methyl moieties [28]. A signal that represented the
methylene protons on the astaxanthin fatty acid moiety
was shown in the middle of 1.307 and 1.593ppm signals.
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Biomass Conversion and Biorefinery
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Fig. 2 Concentration of
astaxanthin extracted from
Penaeus japonicus (A) and
Penaeus semisulcatus (B) using
Saccharomyces cerevisiae and
Lactobacillus acidophilus fol-
lowed by solvent extraction with
hexane and acetone at a ratio of
1:1, 1:2, and 1:3
a
b
c
ab a
d
0
1
2
3
4
5
6
7
8
9
10
123
Astaxanthin (mg/g)
Hexane/Acetone
S cerevisae
L.acidophilus
b
c
e
a
d
de
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1:1 1:
21
:3
Astaxanthin (mg/g)
Hexane/Acetone
S cerevisae
L.acidophilus
A
B
Table 1 Astaxanthin concentrations obtained for the different extracts by using the HPLC method
Shrimp species Extraction method Average peak area [Astaxan-
thin] (µg/
mL)
[Astaxanthin]
(mg/g waste)
Penaeus semisulcatus Lactobacillus acidophilus + solvent extraction with hexane and acetone
at 1:1
307.55 4.88 0.488
Chemical method 253.77 3.774 0.3774
Saccharomyces cerevisiae + solvent extraction with hexane and acetone
at 1:1
168.09 2.0119 0.2012
Penaeus japonicus Lactobacillus acidophilus + solvent extraction with hexane and acetone
at 1:1
443.83 7.68 0.768
Chemical method 501.61 8.87 0.887
Saccharomyces cerevisiae + solvent extraction with hexane and acetone
at 1:1
517.05 9.192 0.9192
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Biomass Conversion and Biorefinery
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Overlapping peaks presented around 1.830 and 2.022ppm
referred to protons of the methylene moiety. A broad sig-
nal found at 3.892ppm corresponded to the OH moiety on
the astaxanthin molecule [29].
4 Discussion
In the marine crustaceans, astaxanthin is considered as the
main carotenoid [30]. Traditionally, chemical extraction of
astaxanthin was being used in the industry. In this study,
the astaxanthin extracted from P. japonicus and P. semi-
culcatus via the chemical method were 8.4 and 3.2mg/g
waste, respectively. These values are comparable to those
attained using the microbiological method, followed by
solvent extraction with hexane and acetone (1:1). How-
ever, Khanafari etal. [20] found that the microbiological
method led to higher astaxanthin yields than the chemical
method for shrimp waste of P. semisulcatus.
The results obtained indicated that astaxanthin extrac-
tion via the microbiological method depended on the
microbiological strain used, as well as the solvent system
used afterwards. Thus, in the present study, P. japonicus
led to a markedly higher astaxanthin yield than P. semisul-
catus. Similarly, Lim etal. [31] elucidated that P. japoni-
cus was a good source of astaxanthin. As for the solvent
system used, increasing the amount of acetone (polar
solvent) in the hexane/acetone solvent mixture decreased
astaxanthin extraction. This might be due to an increase
in the polar solvent, which favored the extraction of other
components, thus hampering astaxanthin extraction.
According to the sustainable development goals
(SDGs), combining microbial with chemical extraction
is better for the environment since it reduces the use of
chemicals [32]. In this sense, green technologies, includ-
ing fermentation via probiotic bacteria, have recently been
developed [33]. Microbial extraction is a humble and eco-
friendly method for the extraction of extremely unstable
pigments such as carotenoids [25] due to the action of the
extracellular proteolytic enzymes secreted by the microor-
ganisms [34]. In addition, shrimp waste is extremely per-
ishable. The carotenoid-rich broth obtained by the micro-
biological process can be stored for a long-time span under
normal storage conditions, which is not possible with other
extraction methods [33].
The presence of astaxanthin was confirmed by NMR
analysis. In the same way, Azizan etal. [28] confirmed the
presence of astaxanthin in the hexane extract of Chaetoceros
calcitrans at 1.34ppm, which represented the methylene
protons on the astaxanthin fatty acid moiety, shown in the
middle of 1.307 and 1.593ppm in the NMR chart of the
present study (Supplementary Fig.4).
5 Conclusion
According to the obtained results, P. japonicus led to a sig-
nificantly higher astaxanthin yield than P. semisulcatus,
which means that the former was a better source of asta-
xanthin. In addition, the extraction of astaxanthin from
shrimp wastes via the microbial method led to equal or
slightly higher yields than thoseextracted via the conven-
tional chemical method. Therefore, the usage of chemicals
in extraction can be reduced and replaced by edible micro-
organisms. This paves the way for the exchange of some
chemicals with useful microorganisms commonly found in
nature.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13399- 022- 02984-2.
Author contribution Salwa A. H. Hamdi: conceptualization, result
analysis, methodology, project administration, writing the original
draft, and supervision; Ghadeer M Ghonaim: methodology, visualiza-
tion, result analysis, and writing the original draft; Rana R. El Sayed:
visualization, methodology, result analysis, and writing the original
draft; Susana Rodríguez-Couto: reviewing and editing manuscript;
Mohamed N. Abd El-Ghany: conceptualization, data curation, formal
analysis, investigation, methodology, result analysis, project admin-
istration, writing the original draft, resources, software, supervision,
validation, visualization, writing, review and editing.
Funding Open Access funding provided by LUT University (previ-
ously Lappeenranta University of Technology (LUT)).
Declarations
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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