Ci a ion: Se a, M.; Casas, A.;
Touba o, D.; Ba os, A.N.; Teixei a,
J.A. Mic obial Hyalu onic Acid
P oduc ion: A Re iew. Molecules
2023,28, 2084. h ps://doi.o g/
10.3390/molecules28052084
Academic Edi o s: Kuan-Chen
Cheng and Fei Tao
Recei ed: 30 Janua y 2023
Re ised: 20 Feb ua y 2023
Accep ed: 21 Feb ua y 2023
Published: 23 Feb ua y 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
molecules
Re iew
Mic obial Hyalu onic Acid P oduc ion: A Re iew
Mónica Se a 1,2,3,*, Ana Casas 1, Dua e Touba o 4, Ana No o Ba os 1,5,* and JoséAn ónio Teixei a 2,3
1Mesosys em, Rua da Ig eja Velha 295, 4410-160 Vila No a de Gaia, Po ugal
2CEB-Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057 B aga, Po ugal
3LABBELS–Associa e Labo a o y, Uni e si y o Minho, 4710-057 B aga, Po ugal
4CBA and Facul y o Sciences and Technology, Uni e si y o Azo es, Rua Mãe de Deus No 13,
9500-321 Pon a Delgada, Po ugal
5
Cen e o he Resea ch and Technology o Ag o-En i onmen al and Biological Sciences (CITAB)), Ins i u e o
Inno a ion, Capaci y Building and Sus ainabili y o Ag i-Food P oduc ion (Ino 4Ag o), Uni e si y o
T ás-os-Mon es and Al o Dou o (UTAD), Quin a de P ados, 5000-801 Vila Real, Po ugal
*
Co espondence: p oduc ion.assis an @mesosys em.com (M.S.); aba os@u ad.p o pca@mesosys em.com (A.N.B.)
Abs ac :
Mic obial p oduc ion o hyalu onic acid (HA) is an a ea o esea ch ha has been gaining
a en ion in ecen yea s due o he inc easing demand o his biopolyme o se e al indus ial
applica ions. Hyalu onic acid is a linea , non-sul a ed glycosaminoglycan ha is widely dis ibu ed
in na u e and is mainly composed o epea ing uni s o N-ace ylglucosamine and glucu onic acid. I
has a wide and unique ange o p ope ies such as iscoelas ici y, lub ica ion, and hyd a ion, which
makes i an a ac i e ma e ial o se e al indus ial applica ions such as cosme ics, pha maceu icals,
and medical de ices. This e iew p esen s and discusses he a ailable e men a ion s a egies o
p oduce hyalu onic acid.
Keywo ds: hyalu onic acid; e men a ion; mic obial p oduc ion
1. In oduc ion
Hyalu onic acid (HA) is a na u ally occu ing biopolyme ha is widely dis ibu ed in
na u e. I is a linea , non-sul a ed glycosaminoglycan ha is composed mainly o epea ing
uni s o N-ace ylglucosamine and glucu onic acid linked by
β
- (1-4) and
β
- (1-3) glycosidic
bonds making i s s uc u e ene ge ically s able (Figu e 1) [
1
]. Each disaccha ide p esen s a
molecula weigh (MW) o a ound 400 Da, and a hyalu onic acid chain can be composed o
10,000 disaccha ides, which means a molecula weigh o a ound 4.0 ×103kDa [2,3].
Molecules 2023, 28, x. h ps://doi.o g/10.3390/xxxxx www.mdpi.com/jou nal/molecules
Re iew
Mic obial Hyalu onic Acid P oduc ion: A Re iew
Mónica Se a 1,2,,*, Ana Casas 1, Dua e Touba o 3, Ana No o Ba os 1,4,* and José An ónio Teixei a 2
1 Mesosys em, Rua da Ig eja Velha 295, 4410-160, São Félix da Ma inha, Vila No a de Gaia, Po ugal;
p oduc ion.assis an @mesosys em.com (M.S.); ana@mesosys em.com (A.C.)
2 CEB-Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057 B aga, Po ugal; LABBELS–Associa e
Labo a o y, Uni e si y o Minho, 4710-057 B aga, Po ugal; ja eixei [email protected]
3 CBA and Facul y o Sciences and Technology, Uni e si y o Azo es, Rua Mãe de Deus No 13,
9500-321 Pon a Delgada, Po ugal; dua e.n . [email protected]
4 Cen e o he Resea ch and Technology o Ag o-En i onmen al and Biological Sciences (CITAB)),Ins i u e
o Inno a ion, Capaci y Building and Sus ainabili y o Ag i-Food P oduc ion (Ino 4Ag o), Uni e si y o
T ás-os-Mon es and Al o Dou o (UTAD), Quin a de P ados, 5000-801 Vila Real, Po ugal;
* Co espondence: p oduc ion.assis an @mesosys em.com (M.S.); aba os@u ad.p o pca@mesosys em.com
(A.N.B.)
Abs ac : Mic obial p oduc ion o hyalu onic acid (HA) is an a ea o esea ch ha has been gaining
a en ion in ecen yea s due o he inc easing demand o his biopolyme o se e al indus ial
applica ions. Hyalu onic acid is a linea , non-sul a ed glycosaminoglycan ha is widely dis ibu ed
in na u e and is mainly composed o epea ing uni s o N-ace ylglucosamine and glucu onic acid.
I has a wide and unique ange o p ope ies such as iscoelas ici y, lub ica ion, and hyd a ion,
which makes i an a ac i e ma e ial o se e al indus ial applica ions such as cosme ics, pha ma-
ceu icals, and medical de ices. This e iew p esen s and discusses he a ailable e men a ion s a -
egies o p oduce hyalu onic acid.
Keywo ds: hyalu onic acid; e men a ion; mic obial p oduc ion
1. In oduc ion
Hyalu onic acid (HA) is a na u ally occu ing biopolyme ha is widely dis ibu ed
in na u e. I is a linea , non-sul a ed glycosaminoglycan ha is composed mainly o e-
pea ing uni s o N-ace ylglucosamine and glucu onic acid linked by β- (1-4) and β- (1-3)
glycosidic bonds making i s s uc u e ene ge ically s able (Figu e 1) [1]. Each disaccha ide
p esen s a molecula weigh (MW) o a ound 400 Da, and a hyalu onic acid chain can be
composed o 10,000 disaccha ides, which means a molecula weigh o a ound 4.0 × 103
kDa [2,3].
Figu e 1. Chemical s uc u e o HA adap ed om [1].
Ci a ion: Se a, M.; Casas, A.;
Touba o, D.; Ba os, A.N.; Teixei a,
J.A. Mic obial Hyalu onic Acid P o-
duc ion: A Re iew. Molecules 2023,
28, x. h ps://doi.o g/10.3390/xxxxx
Academic Edi o (s): Kuan-Chen
Cheng and Fei Tao
Recei ed: 30 Janua y 2023
Re ised: 20 Feb ua y 2023
Accep ed: 21 Feb ua y 2023
Published: 22 Feb ua y 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/license
s/by/4.0/).
Figu e 1. Chemical s uc u e o HA adap ed om [1].
Hyalu onic acid plays an impo an ole in li ing o ganisms and is an a ac i e
bioma e ial o di e en applica ions due o i s ea u es, in pa icula mois u izing e en ion
abili y, iscoelas ici y, esis ance o mechanical damage, and lack o immunogenici y and
Molecules 2023,28, 2084. h ps://doi.o g/10.3390/molecules28052084 h ps://www.mdpi.com/jou nal/molecules
Molecules 2023,28, 2084 2 o 16
oxici y. In many cases, i ac s as a lub ican (join s), a s uc u e s abilize , an o gan space
ille (skin), and a shock abso be (ca ilage) [4–6].
I is a na u ally occu ing polysaccha ide ound in he body ha plays a i al ole
in skin heal h and beau y. I is a majo componen o he ex acellula ma ix (ECM)
and is esponsible o main aining skin hyd a ion, elas ici y, and olume [
5
,
7
]. As we
age, he le els o hyalu onic acid in ou skin dec ease, leading o w inkles, d yness, and
loss o i mness. The e o e, he use o p oduc s con aining hyalu onic acid has become
inc easingly popula in he cosme ic indus y o comba he signs o ageing and p omo e
heal hy, you h ul-looking skin [8].
This biocompa ible polyme immobilizes he wa e in he issue, and i can change
he de mal olume ha in luences cell p oli e a ion, di e en ia ion, and issue epai . The
biological unc ions depend on i s molecula weigh (MW), o example, mucoadhe ence
is a p ope y o hyalu onic acid wi h a high molecula weigh . This ype o hyalu onic
acid is used as space ille s, an iangiogenic and immunosupp essi e, while medium-size
hyalu onic acid chains a e in ol ed in o ula ion, emb yogenesis, and wound epai . Small
chains o hyalu onic acid ha e in lamma o y, immuno-s imula o y, angiogenic, and an i-
apop o ic p ope ies [3,7,9].
Hyalu onic acid is a humec an , which means i a ac s and e ains mois u e. I can
hold up o 1000 imes i s weigh in wa e , making i an e ec i e ing edien in mois u ize s
and se ums. By keeping he skin hyd a ed, hyalu onic acid helps o plump up he skin and
educe he appea ance o ine lines and w inkles. I also imp o es skin elas ici y, making
i appea i me and mo e you h ul. In addi ion o i s abili y o hyd a e and plump he
skin, hyalu onic acid has an i-in lamma o y p ope ies ha can help o educe edness and
i i a ion. This makes i a g ea ing edien o sensi i e skin ypes, as well as o hose wi h
condi ions such as osacea o eczema [8,10].
The applica ions o hyalu onic acid a e di e se such as he co ec ion o acial olds
and w inkles, body con ou ing, and as a ma ke in he diagnosis o umou s. Hyalu onic
acid can also be used in he supplemen a ion o join luid, in eye su ge y, egene a ion o
su gical wounds, and as a d ug deli e y agen o a ious adminis a ion ou es [
2
,
4
,
11
,
12
].
Hyalu onic acid can also be used o enhance he e ec s o o he skinca e ing edien s. Fo
example, when combined wi h e inoids, i can help o educe he d yness and i i a ion ha can
be caused by hese powe ul an i-ageing ing edien s [
13
]. Simila ly, when used in combina ion
wi h an ioxidan s, i can help o p o ec he skin om en i onmen al damage [10,14].
While hyalu onic acid is a na u ally occu ing subs ance in he body, opical p oduc s
con aining hyalu onic acid a e ypically de i ed om ei he oos e combs o e men a ion.
The e a e di e en ypes o hyalu onic acid, such as sodium hyalu ona e and hyalu onic
acid. Sodium hyalu ona e is a sal o m o hyalu onic acid ha is smalle and can pene a e
deepe in o he skin [15].
The demand and he alue o hyalu onic acid ha e inc eased o e he yea s. Acco ding
o R. G. Fe ei a e al. [
16
], he es ima ed ma ke o hyalu onic acid was EUR 7.6 billion in 2019.
I is expec ed o ha e an annual g ow h o 8.1% om 2016 o 2027, which means ha 20 MT o
hyalu onic acid p oduced by ha yea will ha e an a e age p ice be ween EUR 1500/kg and
EUR 4000/kg. The main ma ke segmen s a e de ma ille s in cosme ics, os eoa h i is, and
oph halmology. Consequen ly, i is necessa y o unde s and he imp o emen poin s in he
hyalu onic acid p oduc ion p ocess, including downs eam p ocessing [16].
2. Hyalu onic Acid P oduc ion Using he Fe men a ion P ocess
Mic obial p oduc ion o hyalu onic acid is an a ea o esea ch ha has been gaining
a en ion in ecen yea s. Hyalu onic acid is widely dis ibu ed in na u e [
1
], and i has a
wide ange o p ope ies such as iscoelas ici y, lub ica ion, and hyd a ion ha make i an
a ac i e ma e ial o a ious indus ial applica ions [2,4,11,12].
Mic oo ganisms such as bac e ia and yeas ha e been used o p oduce HA h ough
e men a ion. S ep ococcus zooepidemicus, a G am-posi i e bac e ia, is one o he mos
Molecules 2023,28, 2084 3 o 16
widely used o ganisms o he p oduc ion o HA due o i s high hyalu onic acid p oduc ion
a e and ease o cul i a ion [5,7,17].
T adi ionally, hyalu onic acid is ex ac ed om animal sou ces such as oos e combs
and cocks’ combs. Howe e , he di icul y in con olling animal issue, high cos s, and e hi-
cal conce ns associa ed wi h animal-de i ed HA ha e led o he de elopmen o mic obial
p oduc ion me hods [5,7,9].
Du ing he p oduc ion o hyalu onic acid, he e a e s ill challenges o be o e come,
such as he limi ed p oduc ion o hyalu onic acid due o he high iscosi y o he b o h,
causing di icul ies in he mixing and mass ans e a e o oxygen; compe i ion o he
same p ecu so s o cell g ow h and hyalu onic acid p oduc ion; and he accumula ion o
lac ic acid, he main by-p oduc o HA e men a ion, causing he inhibi ion o cell g ow h
and hyalu onic acid p oduc ion [5,7,9].
To o e come hese challenges and also inc ease he mic obial p oduc ion o hyalu onic
acid, se e al s a egies ha e been s udied: selec ion o p oducing s ains and app op i-
a e cul u e media, he es ablishmen o cul u e me hods, and de e mina ion o cul u e
condi ions, among o he s.
3. Selec ion o a Mic oo ganism P oduce and I s Cul i a ion Media
The selec ion o he app op ia e mic oo ganism o p oduce hyalu onic acid is an
impo an ac o in he mic obial p oduc ion p ocess. Each mic oo ganism has i s own
unique ad an ages and disad an ages in e ms o p oduc ion pe o mance and p o i abili y.
When choosing a mic oo ganism o p oduce hyalu onic acid, i is essen ial o conside he
speci ic equi emen s o he inal applica ion o his molecule [5,7,9].
The mic oo ganism mus be able o p oduce high yields o hyalu onic acid, be easy
o cul i a e, and ha e a low cos o p oduc ion. Se e al mic oo ganisms ha e been used
o p oduce hyalu onic acid h ough e men a ion, including S ep ococcus zooepidemicus,
Bacillus sub ilis, and Esche ichia coli [18–21].
S ep ococcus zooepidemicus p oduces a high molecula weigh HA wi h excellen bio-
compa ibili y, making i sui able o medical applica ions such as wound healing and join
lub ica ion. Howe e , S ep ococcus is a slow-g owing o ganism, and he p oduc ion p o-
cess is cos ly due o he need o expensi e media and downs eam p ocessing. Howe e ,
i does no equi e he use o oxic chemicals o sol en s, esul ing in a pu e and sa e
end p oduc [16,22].
Bacillus sub ilis is ano he mic oo ganism ha has been used o p oduce hyalu onic
acid. I is a G am-posi i e bac e ium ha is known o i s abili y o p oduce high yields o
HA and is also non-pa hogenic. Howe e , i is conside ed mo e di icul o cul i a e han
S ep ococcus zooepidemicus. Bacillus p oduces a lowe molecula weigh hyalu onic acid
ha is sui able o cosme ic applica ions such as mois u izing c eams and se ums. Bacillus
g ows apidly and is ela i ely cheap o p oduce, making i a mo e p o i able op ion o
cosme ic companies [18,23].
Esche ichia coli is a G am-nega i e bac e ium ha has been used o he p oduc ion
o hyalu onic acid. I is a well-s udied o ganism and has a wide ange o gene ic ools
a ailable o use in gene ic enginee ing. Howe e , i p oduces a lowe yield o hyalu onic
acid compa ed o S ep ococcus zooepidemicus and Bacillus sub ilis [20,21,24].
Ano he mic oo ganism ha has been ecen ly explo ed o hyalu onic acid p o-
duc ion is he yeas Pichia pas o is. This o ganism p oduces a high yield o low molec-
ula weigh HA, making i sui able o use in he pha maceu ical indus y. The p o-
duc ion p ocess is ela i ely cheap and can be scaled up easily, making i a p omising
candida e o la ge-scale p oduc ion [25,26].
When conside ing he p o i abili y and possible applica ions o he hyalu onic acid
p oduced by each mic oo ganism, i is essen ial o conside ac o s such as p oduc ion cos s,
pu i y o he inal p oduc , and ma ke demand. Fo example, he high-molecula -weigh
hyalu onic acid p oduced by S ep ococcus has signi ican po en ial in he medical ield
due o i s excellen biocompa ibili y, making i a high- alue p oduc . Meanwhile, he
Molecules 2023,28, 2084 4 o 16
lowe -molecula -weigh hyalu onic acid p oduced by Bacillus has excellen po en ial in he
cosme ic indus y, wi h demand o hyalu onic acid-based cosme ics con inuing o g ow.
In conclusion, he selec ion o he app op ia e mic oo ganism o hyalu onic acid
p oduc ion is c i ical o de e mining he p oduc ion pe o mance and p o i abili y o he
p ocess. By e alua ing he ad an ages o he inal applica ion, p oduce s can op imize hei
p oduc ion p ocess, enhance p oduc quali y, and inc ease p o i abili y.
G oup A and C s ep ococci, namely S ep ococcus zooepidemicus, ha e been he mos
explo ed s ain in he p oduc ion p ocess o mic obial hyalu onic acid and ha e ob ained
he bes esul s. Ne e heless, due o he pa hogenici y o his na u al HA-p oduce
bac e ia, o he me abolically enginee ed mic oo ganisms ha e been s udied, such as Bacillus
sub ilis [
18
,
19
,
23
], Co ynebac e ium glu amicum [
27
–
29
], Esche ichia Coli [
20
,
21
,
24
], Lac ococcus
lac is [
30
,
31
], Pichia pas o is [
25
], and Kluy e omyces lac is [
32
]. Among he ci ed s ains,
Co ynebac e ium glu amicum has p esen ed he bes esul s [28,29].
The cul i a ion medium o p oduce hyalu onic acid A is also an impo an ac o o
conside . The medium mus con ain a sou ce o ca bon, ni ogen, i amins, mine als, and
o he g ow h ac o s o he mic oo ganism. Addi ionally, he medium should be op imized
o p omo e he p oduc ion o hyalu onic acid by he mic oo ganism [5,26].
Conce ning media composi ion o hyalu onic acid p oduc ion, an impo an ac o is
he cul i a ion condi ion. S ep ococcus zooepidemicus is a nu i ionally demanding mic oo -
ganism, and a ni ogen sou ce is an essen ial nu ien o i s g ow h. This bac e ium does
no syn hesize some amino acids ha a e a ou able o i s g ow h and hyalu onic acid
p oduc ion. The e o e, media supplemen a ion wi h nu ien s, such as amino acids, has
been s udied [5,26].
3.1. Al e na i e Sou ces as Subs a es o he Cul u e Media
One o he key ac o s in he p oduc ion o hyalu onic acid is he cul u e medium ha
is used o g ow he mic oo ganism p oduce s.
T adi ionally, he cul u e medium o hyalu onic acid p oduc ion has been based on
complex suga s such as glucose and uc ose, bu hese can be expensi e and may no
be eadily a ailable [
33
,
34
]. As a esul , esea che s ha e been in es iga ing al e na i e
sou ces as subs a es o he cul u e medium. Some o he mos p omising al e na i e
sou ces include:
1.
Ag icul u al was e: By-p oduc s o ag icul u e, such as suga bee pulp, co n s eep
liquo , and whea b an, ha e been ound o be sui able subs a es o hyalu onic acid
p oduc ion. These was e p oduc s a e eadily a ailable and ha e he added bene i o
being en i onmen ally iendly [35].
2.
Indus ial was e: Indus ial was e p oduc s, such as dis ille y was e and molasses,
ha e also been ound o be sui able subs a es o hyalu onic acid p oduc ion. These
was e p oduc s a e o en cheape han adi ional subs a es, making hem an a ac-
i e op ion o comme cial p oduc ion [35,36].
3.
Syn he ic subs a es: Some esea che s ha e also in es iga ed syn he ic subs a es,
such as hyd olysa es o s a ch and cellulose, as an al e na i e subs a e o hyalu onic
acid p oduc ion. These subs a es a e ela i ely cheap and easy o p oduce, making
hem an a ac i e op ion o comme cial p oduc ion [37].
O e all, al e na i e sou ces as subs a es o he cul u e media o hyalu onic acid p o-
duc ion ha e been explo ed o educe he cos s o p oduc ion and make he p ocess mo e
sus ainable. While mo e esea ch is needed o ully unde s and he po en ial o hese al e na i e
sou ces, hey o e p omising possibili ies o he u u e o hyalu onic acid p oduc ion.
Di e en ag icul u al esou ces and indus ial was es ha e been explo ed as al e na i e
nu i i e sou ces o mic obial hyalu onic acid. The goal o he o mula ion o cos -e ec i e
cul u e media is o main ain low cos s du ing mic obial hyalu onic acid p oduc ion and o
educe pollu ion p oblems, as well as o imp o e he e iciency o he e men a ion p ocesses.
Amado e al. [
36
] s udied he op imiza ion o a media con aining cheese whey o
p oduce hyalu onic acid by S ep ococcus zooepidemicus. The majo nu ien s in cheese whey
Molecules 2023,28, 2084 5 o 16
a e lac ose, soluble p o eins (
β
-lac oglobulin,
α
-lac albumin), lipids, and B-g oup i amins.
Using cheese whey p o ein and glucose as ni ogen and ca bon sou ces, espec i ely, he
maximum p oduc ion a e was 0.87 g/L h (0.75 g/L h in con ol media wi h glucose and
yeas ex ac ), he HA p oduc ion was 4.02 g/L (3.19 g/L in con ol media), and he HA
a e age molecula weigh s (HA-MW) we e 3.71 ×103kDa.
In ano he s udy, Amado e al. [
35
] o mula ed a cul u e media con aining co n s eep
liquo (CSL) ins ead o yp one as a ni ogen sou ce. The highes hyalu onic acid p o-
duc ion in his media was 3,48 g/L (compa able o he con ol media o 3.60 g/L) wi h a
molecula weigh o 3.8 ×103kDa (highe han he con ol media o 3.0 ×103kDa).
A slan & Aydogan [
38
] es ed he e ec i eness o sheep wool pep one (SWP) and
molasses as ni ogen and ca bon sou ces, espec i ely, in e men a ion media o p oduce
hyalu onic acid, using he same s ain as Amado e al. They e i ied a highe HA p oduc ion
in media con aining sheep wool pep one (3.54 g/L) han in media con aining yp one TP
(2.58 g/L) and pep one PP (2.47 g/L). Sheep wool pep one has a lowe p o ein con en
(70.6 g/100 g) han yp one and pep one (83.1 and 83.3 g/100 g, espec i ely), and by
con as , sheep wool pep one has highe elemen con en s (K, P, and Mg) han yp one and
pep one, and hese elemen s p omo e hyalu onic acid p oduc ion. Mo eo e , his ni ogen
sou ce con ains high cys ine and a ginine con en s, which a e he main amino acids ha
a ec hyalu onic acid p oduc ion. These a e possible easons o he highe p oduc ion o
hyalu onic acid in sheep wool pep one han in yp one and pep one.
Simila ly, Pan e al. [
39
] de eloped a media o hyalu onic acid p oduc ion which
con ains suga cane molasses. A e molasses ea men wi h ac i a ed cha coal and using
i as a subs a e o S ep ococcus zooepidemicus, he hyalu onic acid p oduc ion was 19%
highe han using molasses wi hou ea men . This sugges ed p e- ea men dec eased
he excessi e me al ions con en ha can inhibi hyalu onic acid p oduc ion. A e 24 h o
e men a ion wi h pH con ol (pH 8), he hyalu onic acid p oduc ion was 2.83 g/L.
Pi es e al. [
40
] in es iga ed he use o cashew apple juice in he e men a ion media,
and he HA p oduc ion was 0.89 g/L wi h a molecula weigh o 18.4 kDa.
Vázquez e al. [
41
] explo ed a cul u e media using ma ine by-p oduc s as subs a es
o S ep ococcus zooepidemicus when p oducing mic obial hyalu onic acid. Fi s ly, glycogen
om mussel p ocessing was ewa e (MPW) and una pep one om isce a esidue we e
used as a ca bon sou ce and a p o ein sou ce, espec i ely. In his media, hyalu onic acid
p oduc ion and he cell g ow h a e we e lowe (2.46 g/L and 0.81 g/L/h, espec i ely)
han in comme cial media con aining glucose and yp one (3.07 g/L and 1.32 g/L/h, e-
spec i ely); on he o he hand, he hyalu onic acid p oduced in al e na i e media p esen ed
a highe molecula weigh (2.50
×
10
3
kDa). La e , he same scien is s explo ed a media
o mula ed wi h pep ones ob ained om Scylio hinus canicula isce a by-p oduc s. Using
his al e na i e subs a e as a ni ogen sou ce, hyalu onic acid p oduc ion was explo ed in
ed-ba ch cul u e. Using his cul u e mode, he hyalu onic acid p oduc ion (2.53 g/L) and
molecula weigh (2.11
×
10
3
kDa) o he polysaccha ide we e inc eased compa ed o p o-
duc ion using he ba ch mode (2.26 g/L; 1.80
×
10
3
kDa). Compa ed wi h he comme cial
media (3.23 g/L; 0.930 g/L/h; 1.85
×
10
3
kDa), he hyalu onic acid p oduc ion and cell
g ow h a e we e lowe in he al e na i e media; howe e , he hyalu onic acid molecula
weigh was highe [42].
Benedini & San ana [
43
] s udied he e ec o eplacing B ain Hea In usion (BHI) wi h
soy pep one (SP) as a ni ogen sou ce o S ep ococcus zooepidemicus. In his case, he e was
an inc emen in hyalu onic acid p oduc ion and hyalu onic acid molecula weigh om
0.29 g/L and 3.09
×
10
3
kDa o 0.30 g/L and 3.60
×
10
3
kDa, espec i ely. The inc ease in
hyalu onic acid molecula weigh was ela ed o he diminu ion o lac ic acid p oduc ion
and hyalu onic acid exposi ion o a highe pH.
Ano he s udy was pe o med in which Ghodke e al. [
44
] e alua ed palmy a palm
suga (Pj) and soya pep one o o mula e a media o hyalu onic acid p oduc ion by S ep-
ococcus zooepidemicus. Palmy a palm suga con ains suc ose and i amins (nico inic acid,
hiamine, ibo la in, and i amin C) ha a e essen ial o he g ow h o a mic oo ganism,

Molecules 2023,28, 2084 6 o 16
which led o he hyalu onic acid p oduc ion and speci ic g ow h a e on palmy a palm
suga -based media (0.41 g/L; 0.54 h
−1
) being highe han on pu e suc ose-based media
(0.31 g/L; 0.32 h
−1
). A e explo ing he bes ini ial subs a e concen a ion (Pj, 30 g/L), he
mic oo ganism yielded a hyalu onic acid concen a ion o 1.22 g/L wi h 9.50 ×102kDa.
Zhang e al. [
37
] p oposed a se um- ee s a ch media in which he cells could g ow
and p oduce hyalu onic acid. Replacing glucose wi h s a ch ob ained hyalu onic acid wi h
a yield o 6.7 g/L. The s udied s ain could g ow in se um- ee media bu no in o he
ca bohyd a es such as glucose. This could be due o he educ ion in lac ic acid (me aboli e
om glucose ca abolism) in he b o h o dec easing glucose in he media.
In hei wo k, Du eck e al. [
45
] op imized he p oduc ion o hyalu onic acid in suga -
cane molasses p e- ea ed wi h ac i e cha coal media (likewise explo ed by Pan e al. [
39
])
wi h nu ien supplemen a ion (glu amine). They concluded ha he hyalu onic acid p o-
duc ion by S ep ococcus zooepidemicus was highe in suga cane molasses media (0.710 g/L)
han in glucose media (0.469 g/L) due o he molasses media being ich in suga and amino
acids, and a e ea men , his molasses con ains ewe inhibi o s o hyalu onic acid p o-
duc ion. Rega ding media supplemen a ion, glu amine supplemen a ion e ealed posi i e
e ec s on hyalu onic acid p oduc ion, imp o ing he yield o he p oduc o 2.55 g/L,
because his amino acid ac s as he amino dono g oup o UDP-N-ace ylglucosamine
o ma ion (one o hyalu onic acid p ecu so s).
3.2. Supplemen a ion o Cul u e Media
The p ocess o media supplemen a ion in he p oduc ion o hyalu onic acid includes:
1.
Ca bon sou ce supplemen s: Ca bon sou ces such as glucose, uc ose, and mal ose
can be added o he cul u e medium o p o ide an addi ional ene gy sou ce o he
mic oo ganisms. This can help inc ease he a e o hyalu onic acid p oduc ion [46].
2.
Ni ogen sou ce supplemen s: Ni ogen sou ces such as ammonium sul a e and yeas
ex ac can be added o he cul u e medium o p o ide essen ial amino acids and
o he nu ien s o he mic oo ganisms. This can help inc ease he a e o hyalu onic
acid p oduc ion [33].
3.
Vi amin supplemen s: Vi amins such as hiamine, ibo la in, and py idoxine can be
added o he cul u e medium o p omo e he g ow h o mic oo ganisms and inc ease
he a e o hyalu onic acid p oduc ion [47].
4.
Mine al supplemen s: Mine als such as sodium, po assium, and calcium can be added
o he cul u e medium o p o ide essen ial mine als o he mic oo ganisms and
p omo e hyalu onic acid p oduc ion [34].
5.
G ow h ac o s: G ow h ac o s such as yeas ex ac , soy pep one, and yp one can
be added o he cul u e medium o p o ide addi ional nu ien s and g ow h ac o s o
he mic oo ganisms [
33
]. This can help inc ease he a e o hyalu onic acid p oduc ion.
I is impo an o no e ha he app op ia e supplemen s and he op imal concen a ion
o he supplemen in he cul u e medium can a y depending on he mic oo ganism
used o hyalu onic acid p oduc ion. Resea che s o en use a combina ion o di e en
supplemen s and weak he concen a ion o ind he bes condi ion o p oduce hyalu onic
acid. I is also impo an o no e ha he use o supplemen s in he cul u e medium can
inc ease p oduc ion cos s; hence, i is impo an o ind a balance be ween cos s and he
enhancemen o p oduc ion.
The subs a es used mo e o en in hyalu onic acid p oduc ion a e glucose and su-
c ose as he p ima y ca bon and ene gy sou ces and yeas ex ac and pep ones as ni-
ogen sou ces [
33
,
34
,
48
–
52
]. O he ca bon sou ces ha e been explo ed as subs a es o
hyalu onic acid p oduc ion, namely, lac ose, mal ose, galac ose, mannose, and o he s;
howe e , hyalu onic acid p oduc ion is no as high [18,34,53–55].
In hei in es iga ion, Chen e al. [
56
] used glucose as a ca bon sou ce and yeas ex ac
as a ni ogen sou ce o explo e he bes ca bon- o-ni ogen (C/N) a io and concluded ha he
C/N a io o 2:1 maximizes hyalu onic acid syn hesis a 2.45 g/L. In o he C/N a ios, such as
1,3:1 and 4:1, he hyalu onic acid p oduc ion was 2.20 g/L and 1.52 g/L, espec i ely.
Molecules 2023,28, 2084 7 o 16
Conce ning media supplemen a ion, A oska e al. [
46
] also in es iga ed he e ec o
di e en nu ien s in he cul u e media o S ep ococcus zooepidemicus p oduc ion o hyalu onic
acid. In his s udy, he ca bon sou ce used was dex ose o p oducing hyalu onic acid wi h a
highe yield (0.70 g/L) han o he ca bon sou ces used, such as suc ose (0.51 g/L), mal ose
(0.50 g/L), and dex in (0.55 g/L). The esea che s explo ed he addi ion o L-a ginine HCl
and annic acid o he e men a ion media, which esul ed in an inc emen in he yield o HA
o 1.029 g/L. L-a ginine HCl is a ca bon and ni ogen dono o he syn hesis o nucleo ides
ha a e needed o he g ow h and mul iplica ion o mic oo ganisms, hus sa ing ene gy
consump ion in he o ganism o i s p oduc ion. Tannic acid ep esses he syn hesis o he
hyalu onidase enzyme ha ac s by depolyme izing hyalu onic acid. In ligh o his, he use o
annic acid showed an inc ease in he molecula weigh o hyalu onic acid.
In he same way, Shah e al. [
33
] explo ed he supplemen a ion media o S ep ococcus
zooepidemicus wi h glu amine and sodium iodoace a e. Iodoace a e dec eases lac ic acid
syn hesis by inhibi ing he glycolysis pa hway; consequen ly, he e was a edi ec ion o
he ca bon low om lac ic acid o ma ion o UDP glucu onic acid o ma ion, one o he
p ecu so s o hyalu onic acid. Glu amine, as men ioned abo e, is an amino acid in ol ed in
he o ma ion o one o he hyalu onic acid p ecu so s. Acco dingly, he HA concen a ion
was inc eased om 2 g/L in media wi hou supplemen a ion o 5 g/L, he speci ic g ow h
a e was dec eased om 0.42 h
−1
o 0.25 h
−1
, and he hyalu onic acid molecula weigh
was inc eased om 2.4 ×103kDa o 3.2 ×103kDa.
Aiming o ob ain HA wi h high yield and molecula weigh , Im e al. [
34
] s udied
he op imiza ion o media componen s using he s ain S ep ococcus sp. ID9102 (KCTC
11935BP), which is nega i e o hemoly ic ac i i y and hyalu onidase. Explo ing he bes
ca bon sou ce, a highe hyalu onic acid p oduc ion was eached when glucose was he
ca bon sou ce (1.58 g/L) ollowed by lac ose and suc ose, which eached simila esul s
(
≈
1.3 g/L). Acco ding o he esul s, he in es iga o s selec ed he bes componen s media
as glucose, yeas ex ac , casein pep one, K
2
HPO
4
, and MgCl
2
and ob ained a hyalu onic
acid p oduc ion o 5.88 g/L wi h a molecula weigh o 3.60
×
10
3
kDa. A e supplemen ing
he media wi h amino acids (glu amine and glu ama e) and o ganic acid (oxalic acid), he
HA i e and HA-MW we e highe (6.94 g/L and 5.90 ×103kDa, espec i ely).
Kim e al. [
47
] also s udied he p oduc ion o HA om S ep ococcus equi mu an
KFCC 10830. This mu an has nonhemoly ic and hyalu onidase-nega i e cha ac e is ics.
Fu he mo e, he scien is s explo ed he e ec o lysozyme addi ion du ing cul i a ion on
he e men a ion media. Lysozyme can damage he cell walls o hyalu onic acid p oduce
mic oo ganisms, which hen p oduce mo e hyalu onic acid o p o ec hemsel es om he
ac ion o lysozyme. Acco ding o his, he addi ion o lysozyme inc eased he p oduc ion
and molecula weigh o hyalu onic acid.
4. De e mina ion o Cul u e Condi ions
Ano he impo an ac o o de elop a success ul p ocess o p oducing mic obial
hyalu onic acid is iden i ying ideal e men a ion pa ame e s.
The cul u e condi ions e e o he a ious ac o s ha can a ec he g ow h and
p oduc ion o he mic oo ganisms used in hyalu onic acid p oduc ion. Some o he key
cul u e condi ions ha mus be de e mined include:
1.
Tempe a u e: The op imal empe a u e o he g ow h o he mic oo ganisms and he
p oduc ion o hyalu onic acid will a y depending on he species used. The empe a u e
ange ha is mos sui able o p oduce HA is usually be ween 30 and 37 ◦C [50].
2.
pH: The pH o he cul u e medium is ano he impo an cul u e condi ion ha mus
be de e mined. The pH mus be main ained wi hin a speci ic ange ha is sui able o
he g ow h o he mic oo ganisms and he p oduc ion o hyalu onic acid. The op imal
pH ange o HA p oduc ion is usually be ween 6.5 and 7.5 [57,58].
3.
Ae a ion: Adequa e ae a ion is necessa y o he g ow h o he mic oo ganisms and
he p oduc ion o hyalu onic acid. The amoun o ai supplied o he cul u e medium
can be adjus ed o achie e he op imal ae a ion o he mic oo ganisms used [59].
Molecules 2023,28, 2084 8 o 16
4.
Agi a ion: Agi a ion is he p ocess o physically s i ing he cul u e medium o
ensu e ha he mic oo ganisms a e well-mixed and ha e access o nu ien s and
oxygen. The op imal agi a ion a e will depend on he mic oo ganisms used and
he cul u e medium [49].
5.
Subs a e concen a ion: The concen a ion o he subs a e in he cul u e medium
will a ec he a e o hyalu onic acid p oduc ion. The op imal subs a e concen a ion
will depend on he mic oo ganisms used and he cul u e medium [51].
6.
Supplemen a ion: Supplemen a ion o he cul u e medium wi h a ious nu ien s
and g ow h ac o s can also a ec he a e o hyalu onic acid p oduc ion. The
op imal concen a ion o supplemen s will depend on he mic oo ganisms used
and he cul u e medium [48,49,60].
De e mining he op imal cul u e condi ions o p oduce hyalu onic acid will ypically
in ol e a p ocess o expe imen a ion and op imiza ion. Resea che s will o en use a
combina ion o di e en cul u e condi ions and weak he a ious ac o s o ind he
condi ions ha yield he highes hyalu onic acid p oduc ion [57–61].
In hei wo k, Johns e al. [
57
] iden i ied he op imum pH as 6.7. A high agi a ion
a e o 600 pm was ound o be ad an ageous o elease he HA capsule om S ep-
ococcus zooepidemicus bac e ia cells in o he medium; howe e , a highe shea a e can
damage he polyme . In addi ion, he use o ae a ed cul u es enhanced he hyalu onic acid
concen a ion, maybe due o he highes ene gy yield.
Da id C. A ms ong & Johns [
50
] desc ibed he e ec o di e en cul u e condi ions
on he molecula weigh o HA p oduced by S ep ococcus zooepidemicus. Conce ning em-
pe a u e, hei esul s showed ha lowe empe a u es (32
◦
C) esul ed in highe molecula
weigh and highe yield o he p oduced hyalu onic acid han highe empe a u es (a
40 ◦C
: 1.85
×
10
3
kDa and 1.20 g/L, espec i ely), and by dec easing he empe a u e, he
speci ic g ow h a e also dec eased, as he wo p ocesses (cell g ow h and HA p oduc ion)
a e compe i o s o he same p ecu so s. Thei esul s also showed ha highe ae a ion
a es (1.0 m) enhanced HA p oduc ion and hyalu onic acid molecula weigh om
1.45 g/L
and 2.10
×
10
3
kDa (0.2 m) o 4.20 g/L and 3.0
×
10
3
kDa, espec i ely. F om
he o he cul u e condi ions explo ed, he ini ial glucose concen a ion also p esen ed an
in luence on hyalu onic acid p oduc ion. A highe ini ial glucose concen a ion (60 g/L) in
he e men a ion b o h enhanced he HA concen a ion and HA-MW, which could be due
o HA p ecu so s (UDP-GlcUA and UDP-GlcNAc) being de i ed om glucose. Howe e ,
he ini ial glucose concen a ion is limi ed o 60 g/L because a highe concen a ions, he e
a e limi a ions in mass ans e due o he high b o h iscosi y.
Simila ly, Don & Shopa we [
51
] explo ed he e ec o glucose in he e men a ion
media o S. zooepidemicus on HA p oduc ion. The highes hyalu onic acid molecula weigh
(2.52
×
10
3
kDa) was eached when he ini ial glucose concen a ion was 40 g/L. Abo e
hese concen a ion le els, he e was a educ ion in hyalu onic acid molecula weigh .
To achie e he highes concen a ion o hyalu onic acid (5.3 g/L), Zake i & Rasaee [
52
]
iden i ied he bes e men a ion condi ions. This way, a e hey explo ed a ious e men a-
ion condi ions, hey p oposed HA p oduc ion wi h he ollowing condi ions: 37
◦
C, pH 7,
300 pm, and DO (dissol ed oxygen) 50%. Mo eo e , hey also iden i ied he bes sou ces
o ca bon and ni ogen: glucose (70 g/L) and yeas ex ac (30 g/L).
Using he same subs a es as Zake i & Rasaee [
52
], Huang e al. [
59
] explo ed he
ole o dissol ed oxygen and he e ec o agi a ion on hyalu onic acid p oduc ion using
e men a ion. They ound an inc emen in hyalu onic acid yield ( om 2.7 g/L o 3.1 g/L)
when dissol ed oxygen inc eased om 2.5% o 5%, and abo e hese alues o dissol ed
oxygen, he HA yield was no in luenced. So, he in es iga o s ound a c i ical le el o
dissol ed oxygen o p oduce HA, and hey sugges ed dissol ed oxygen o ha e a s imulan
ole in hyalu onic acid syn hesis. Conce ning agi a ion, i s main unc ion is o mix he b o h;
howe e , agi a ion can a ou oxygen abso p ion by cells. A 400 pm (
0.74 g HA/g cell
),
he hyalu onic acid p oduc i i y was highe han a 200 pm (0.65 g HA/g cell).
Molecules 2023,28, 2084 9 o 16
Unde ae obic condi ions a 37
◦
C and wi h 200 pm agi a ion, L Liu e al. [
58
] explo ed
an in e mi en alkaline-s ess s a egy. Unde s ess condi ions a pH 8.5 o 1 h o 6 h o
e men a ion, a hyalu onic acid concen a ion o 6.5 g/L was ob ained (highe han con ol
condi ions: 5.0 g/L). This inc ease in p oduc i i y may be due o he edi ec ion o he
ca bon low (mo e a ailabili y o ca bon sou ce o p oduce hyalu onic acid a he han cell
g ow h o lac ic acid p oduc ion) and/o he inc ease in dissol ed oxygen ha was ound
in s ess condi ions.
Oxygen mass ans e has an impo an ole in hyalu onic acid p oduc ion. None he-
less, he high iscosi y o he media caused by hyalu onic acid p oduc ion can chal-
lenge ideal mixing and uni o m oxygena ion. To o e come his p oblem, Saha khiz &
Babaeipou [
60
] s udied he e ec o dilu ing he cul u e media con olling pH wi h a
low concen a ion o NaOH. The dilu ion imp o ed he mass ans e o oxygen, and he
hyalu onic acid p oduc ion was enhanced om 6.6 g/L o 8.4 g/L.
Long Liu e al. [
49
] p oposed h ee s a egies o con ol dissol ed oxygen in he
p oduc ion media. Fi s ly, hey applied a h ee-s age agi a ion speed con ol cul u e model
in which hey a ied he agi a ion speed be ween 200 pm (0–8 h), 400 pm (8–12 h), and
600 pm (12–20 h)
. The HA p oduc i i y was enhanced om 5.0 g/L a cons an agi a ion
speed (200 pm) o 5.5 g/L. This may be due o he inc ease in he dissol ed oxygen le el
ha is a ou able o he g ow h o he cells, and he inc ease in agi a ion ha induces
shea s ess o cells. Following he p e ious model, he in es iga o s explo ed a wo-s age
dissol ed oxygen con ol model. In his model, hey con olled he dissol ed oxygen le el
a he 10% le el du ing he i s 8 h o he e men a ion p ocess, and a e , hey dec eased
he dissol ed oxygen le el o 5% using agi a ion speed con ol. Once he e was a di e ence
in he c i ical dissol ed oxygen le els o cell g ow h and hyalu onic acid p oduc ion, wi h
his s a egy, i was possible o edi ec he ca bon low in he i s phase o he e men a ion
p ocess o cell g ow h and, la e , o he p oduc ion o hyalu onic acid. As a esul , he
HA p oduc ion and p oduc i i y inc eased o 6.3 g/L and 0.0984 g/L/h ( om 5 g/L and
0.0694 g/L/h
, espec i ely). Ano he s a egy o imp o e dissol ed oxygen le els in he
b o h is media supplemen a ion wi h oxygen ec o s. In his way, Long Liu e al. [
36
]
explo ed he e ec o PFC (pe luo odecalin) addi ion o he media a 8 h o he p ocess. I
was ound ha pe luo odecalin imp o ed he dissol ed oxygen le els om 0.5% o 5% a
a low agi a ion speed (200 pm), and he HA i e in he media was 6.6 g/L.
Likewise, Z. W. Lai e al. [
48
] in es iga ed he e ec o n-hexadecane as an oxygen
ec o . This oxygen ec o enhanced he hyalu onic acid concen a ion om 2.45 g/L o
4.25 g/L and he hyalu onic acid molecula weigh om 5.20
×
10
3
kDa o 1.54
×
10
4
kDa.
So, his sugges ed n-hexadecane can be used as an oxygen ec o and o ganic phase o
inc easing he molecula weigh o hyalu onic acid. This in es iga ion o Lai e al. [
48
]
con i med wha was epo ed in he Duan e al. [
62
] s udies. In hei wo k, hey explo ed he
e ec o dissol ed oxygen le els on he molecula weigh o hyalu onic acid and concluded
ha molecula weigh is dependen on he balance o he e ec o ATP and eac i e oxygen
species (ROS) in he cul u e media. Mo eo e , highe dissol ed oxygen le els enhanced
ATP le els and ROS in he media. As i is known, ATP and ROS le els ha e con a y e ec s,
so he highes HA-MW (2.19
×
10
3
kDa) was eached on he balance o hese wo e ec s a
he 50% dissol ed oxygen le el. Abo e his le el (80% DO), he HA-MW was dec eased
(2.06 ×103kDa).
To achie e a balance be ween a highe hyalu onic acid concen a ion and a highe
hyalu onic acid molecula weigh , J. Liu e al. [
63
] de eloped a wo-s age e men a ion
s a egy. The highes hyalu onic acid concen a ion and hyalu onic acid molecula weigh
we e eached a he same condi ions o ae a ion a e and agi a ion speed (1 m and
600 pm
, espec i ely). Howe e , hese alues o concen a ion and molecula weigh we e
achie ed a di e en condi ions o empe a u e and pH. The i s s age o e men a ion was
pe o med a pH 8 and 31
◦
C allowing o he bes condi ions o molecula weigh g ow h,
and las ly, he inal s age o he p ocess was conduc ed a pH 7 and 37
◦
C o p omo e HA
Molecules 2023,28, 2084 16 o 16
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