Studies on the formation mechanism of zinc protoporphyrin IX from heme proteins via the heme dissociation in nitrite/nitrate-free meat products [an abstract of entire text]

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Studies on the formation mechanism of zinc protoporphyrin IX from heme proteins via the heme dissociation in
nitrite/nitrate-free meat products [an abstract of entire text]

Zhai, Yang

北海道大学. 博士(農学) 甲第15296号

2023-03-23

http://hdl.handle.net/2115/89548

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theses (doctoral - abstract of entire text)

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博 士 論 文 の 要 約

博士の専攻分野の名称

博 士（農学）

氏名

翟 洋 （Zhai Yang）

学 位 論 文 題 名
Studies on the formation mechanism of zinc protoporphyrin IX from heme
proteins via the heme dissociation in nitrite/nitrate-free meat products
亜硝酸塩/硝酸塩無添加食肉製品中におけるヘムの解離を介したヘムタンパク質
からの亜鉛プロトポルフィリン IX の形成機構に関する研究
[Backgrounds and objectives]
Color is one of the most important factors of meat and meat products affecting consumer
buying decisions. In the meat processing industry, synthetic nitrate and nitrite are widely
used in meat products for their color-fixative properties and their inhibitory effects on
bacteria growth, particularly Clostridium botulinum. Nitrate and nitrite are also known for
their antioxidant and anti-rancidity properties, as well as their contribution to the
characteristic cured meat flavor. However, the use of nitrate and nitrite has raised concerns
due to its potential to form carcinogenic nitrosamines. Since the requirements of consumers
for organic and clean-label meat products have increased due to the concerns about the
health risk of synthetic nitrate and nitrite, the meat industry has currently concerned with
the development of their alternatives. Zinc protoporphyrin IX (ZnPP) is a distinct red
pigment in traditional Italian Parma ham (Prosciutto di Parma), that has matured over one
year after curing with only sea salt. In addition, the stable bright red color of Parma ham is
mainly due to the formation of ZnPP. Since the formation of ZnPP is also found in other
nitrite-free dry-cured meat products, such as the dry-cured Iberian hams and the dry-cured
fermented sausages, ZnPP is considered as a potential pigment to improve the color of drycured meat products without adding nitrite and nitrate or any coloring agents.
In recent years, it has been proposed that ZnPP in Parma ham is mainly formed through
the conversion of heme, which comes from the endogenous heme proteins, such as
hemoglobin (Hb) and myoglobin (Mb). After heme is dissociated from heme proteins, it is
first converted into protoporphyrin IX (PPIX) through an iron-removal reaction.
Subsequently, the formation of ZnPP is explained by the insertion of the zinc ion into PPIX.
In addition, although ZnPP itself is water-insoluble, ZnPP mainly exists in Parma ham as
water-soluble complexes by binding with Hb (ZnPP-Hb) and Mb (ZnPP-Mb). However,
the specific formation pathway of these water-soluble ZnPP complexes in Parma ham
remains unclear. Additionally, the formation of ZnPP is proposed to be strongly associated

with Mb owing to its highest content in meat than other heme proteins, such as Hb. Also,
after slaughter, only a small amount of Hb remains in the meat. However, the amount of
ZnPP-Hb is approximately threefold higher than that of ZnPP-Mb in Parma ham, indicating
Hb may be a more crucial hemeprotein in the formation of the water-soluble ZnPP in Parma
ham compared to Mb. Therefore, in the first part of this study, we attempt to elucidate the
formation pathway of the water-soluble ZnPP complex in Parma ham and the mechanism
by which ZnPP-Hb is dominated in Parma ham compared with ZnPP-Mb.
On the other hand, nitrite or/and nitrate, which are added to common cured meat products,
have been found to inhibit the formation of ZnPP in meat. This phenomenon is proposed
as the nitric oxide (NO), which is derived from nitrite, can degrade the [2Fe–2S] cluster of
mammalian FECH, further suppressing its catalytic activity of iron-removal and zinc
insertion to form ZnPP in meat. However, NO as a kind of heme ligand can also strongly
bind to the heme iron of the meat-endogenous heme proteins to form the structurally stable
nitrosyl heme proteins, which bring a long-lasting, bright red color for the nitrite-added
cured meat. This phenomenon indicated that NO derived from nitrite may also inhibit ZnPP
formation by limiting the heme dissociation from the heme proteins in meat. Therefore, the
significance of heme dissociation from heme proteins on ZnPP formation may be
investigated using NO to interact with endogenous heme proteins. Nevertheless, since NO
can bind with either the heme (Fe2+) or the hematin (Fe3+) in heme proteins to make them
structurally stable, it is hard to elucidate the specific pathway of heme or hematin
dissociation from heme proteins to form ZnPP only using NO. However, the carbon
monoxide (CO) can only bind with the heme of heme proteins to form the highly
structurally stable carboxy heme proteins, while the azide ion can bind with the hematin of
the oxidized heme proteins to form the structurally stable azide heme proteins. Besides,
CO and azide ion does not cause disassembly of the FECH [2Fe-2S] cluster in meat, which
is different from that of NO. Therefore, in the second part of this study, we aim to clarify
the significance of heme dissociation from the heme proteins in the formation mechanism
of ZnPP in meat by investigating the inhibitory pathway of NO on ZnPP formation from
heme proteins compared with CO and sodium azide. Additionally, some reductants have
been reported to be essential for the iron-removal reaction of hematin to form ZnPP under
anaerobic conditions, but the over-reduction might inhibit ZnPP formation. Therefore, the
effects of the free heme reduction and the over-reduction on ZnPP formation were also
evaluated in the second part of this study.
1. Investigating the mechanism of the water-soluble ZnPP formation in Parma ham
using a new experimental model
[Materials and Methods]
To investigate the formation mechanism of ZnPP in Parma ham more efficiently and
conveniently, based on previous ZnPP-forming experimental models, a new experimental
model producing plenty of water-soluble ZnPP like Parma ham need to establish first.
Single-factor experiments were carried out to determine the optimum level of each
parameter in the new experimental model (meat content, incubation temperature, pH, and

incubation time) with respect to ZnPP formation. The fluorescent intensity of the model
supernatant was used as a metric for the quantity of water-soluble ZnPP formed, and the
fluorescence intensity of the model precipitate was used as a metric for the quantity of
water-insoluble ZnPP formed. The water-soluble ZnPP complexes formed in the new
experimental model were then verified whether they were the same as those in Parma ham
using SEC-HPLC, urea-PAGE, and western blotting analysis. To clarify how ZnPP-Hb is
dominant over ZnPP-Mb in Parma ham, exogenous Hb or Mb standard was added to the
experimental model at the same final concentration, and the changes in water-soluble ZnPP
and total ZnPP (the sum of water-soluble and water-insoluble ZnPP) were monitored by
measuring their fluorescent intensities during anaerobic incubation. The released non-heme
iron content in the model supernatant was measured using a 1,10-phenanthroline
colorimetric method. The protein stabilities of Hb and Mb standards were evaluated by
measuring the changes in their heme absorbance spectrum and tryptophan (Trp)
fluorescence spectrum after 3 days of anaerobic incubation. Finally, to investigate the
binding reaction of ZnPP with heme proteins to form water-soluble ZnPP complexes, the
apo-form of Hb (Apo-Hb), which was prepared by removing the heme from the Hb, was
mixed with the ZnPP standard, and the mixture was then detected using urea-PAGE.
[Results and discussion]
The optimal condition for the new experimental model was found as incubating 50%
porcine longissimus thoracis et lumborum muscle at pH 5.5 for 10 days at 35℃. Compared
with the previous models, the water-soluble ZnPP in the new model increased seven-fold,
and the water-insoluble ZnPP increased four-fold, indicating that plenty of water-soluble
ZnPP complexes could form in the new experimental model. According to the results of
urea-PAGE, the fluorescent bands of water-soluble ZnPP complexes in the new
experimental model were almost the same as the main fluorescent bands of water-soluble
ZnPP complex in Parma ham, and these bands were detected as ZnPP-Hb using western
blotting. In HPLC results, two peak fractions that related to the water-soluble ZnPP
complexes were observed in both the new experimental model and Parma ham. After
calculating their molecular weights, one peak fraction was considered as Hb dimer and the
other peak fraction was considered as the free ZnPP. These results indicated that ZnPP-Hb
dimer was the main water-soluble ZnPP complex in the new experimental model which
was consistent with that in Parma ham. Therefore, the new experimental model was
proposed to be suitable for investigating the formation mechanism of water-soluble ZnPP
in Parma ham. The addition of the exogenous Mb in the new experimental model had no
effect on the total ZnPP, water-soluble ZnPP, and released non-heme iron increase
compared with those in the control group, in which no heme substrates was added. In
contrast, when exogenous Hb was added into the new experimental model, it significantly
promoted the increase of the total ZnPP, water-soluble ZnPP, and released non-heme iron
compared with both control and Mb-added groups. These results indicated that, compared
with Mb, the heme in Hb was easier to dissociate and convert into ZnPP and water-soluble
ZnPP complexes. ...

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