Recycling of Waste Food by High Pressure Carbon Dioxide Treatment and the Application of Recycled Products( 本文 (Fulltext) )

Title Recycling of Waste Food by High Pressure Carbon Dioxide Treatment and the Application of Recycled Products( 本文 (Fulltext) )

Author(s) YU TONGHUAN

Report No.(Doctoral

Degree) 博士(農学) 甲第773号

Issue Date 2021-09-17

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/82784

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Recycling of Waste Food by High Pressure Carbon Dioxide Treatment and the Application of Recycled Products

（食品ロスリサイクル化プロセスへの炭酸ガス殺菌技術の適用と リサイクルプロダクトの利用）

2021

The United Graduate School of Agricultural Science, Gifu University

Science of Biological Resources (Gifu University)

YU TONGHUAN

Recycling of Waste Food by High Pressure Carbon Dioxide Treatment and the Application of Recycled Products

（食品ロスリサイクル化プロセスへの炭酸ガス殺菌技術の適用と リサイクルプロダクトの利用）

YU TONGHUAN

2

概要

：

2011年から2016年の国連食糧農業機関からの報告によると、毎年の食品ロスと廃棄 物は大量である。これらの食品ロスと廃棄物には、多くの栄養素、特にタンパク質含有 量の高い魚や肉が含まれている。また、生肉・魚類は、動物飼料添加物、微生物培地、

肥料添加物など、他の産業用途の資源として魅力的である。これらを考慮して、高タン パク質の廃肉・魚類を酵素加水分解によってリサイクルすることを試みた。その際、非 加熱殺菌技術として期待さている高圧二酸化炭素（HPCD）殺菌技術を使用すると、酵素 加水分解プロセス中の廃肉・魚類に対する微生物汚染を防ぐ事が可能と考えて本研究を 遂行した。

牛肉を使った実験によると、HPCD は比較的低温での工業的酵素加水分解プロセスに お け る 新 し い 殺菌 技 術 と し て 採 用 さ れる 可 能性 を 示 し た 。 HBI EnzymesInc の

OrientaazeOPが、この研究で最適な工業用プロテアーゼとして選択したところ、1 MPa

16時間 50℃で、牛肉の高度な分解効率を達成できた。HPCD処理加水分解物と非HPCD処 理加水分解物の間で遊離アミノ酸濃度に差は無かった。このことはHPCD技術がバイオ マス分解のための工業的酵素加水分解プロセスに適用できることを示している。すなわ ち、バイオマスが比較的低温で微生物によって汚染を防ぎながら、工業用酵素加水分解 プロセスと組み合わせる事が可能な新しい技術となる可能性がある。牛肉の処理により 得られた加水分解物を培地合成に使用し、E. coliK12JM109およびS.cerevisiaeS288C を培養したところ、充分な生育速度が得られた。

一方、サケは広く消費されており、多種多様なタンパク質と脂質が含まれているが、

食品ロスとして未利用部分の多い資源でもある。資源化を検討したところ、50°C およ

び1 MPaで16時間のHPCD処理で、鮭の微生物汚染を効果的に防止することが確認され

た。HPCD 処理下で加水分解されたサケの安全性を確認するために、この研究では次世 代シーケンシング（NGS）とリアルタイムPCRを使用して、HPCD処理中にサケに存在す る細菌の動的変化を分析した。リアルタイム PCR 分析は、HPCD が中程度の温度でバク テリアの増加を抑制することもできることを示した。 NGSによる分析では、HPCDを使 用して 50℃および 1 MPa で 16 時間処理したサンプルで検出された、

Pseudomonas, Acinetobacter, Enterobacter, Klebsiella,

などのヒスタミン生産菌の存在量は非常に少な いことが判明した。加水分解物分析は、1MPaでのHPCD処理が鮭からの加水分解物に影 響を与えなかったことが分かった。 Orientaaze OPによって生産された鮭からの加水 分解物は、微生物培地、動物飼料添加物、アミノ酸肥料など、商業的価値の高い製品で 使用するための魅力的な生物資源である可能性がある。

これらの研究の結果は、工業的酵素加水分解における HPCDの適用性を支持し、バイ オベースの材料の持続可能性を高めることが期待される。 本研究は、HPCDの下で酵素 反応を使用して、廃肉や廃鮭などの生命の犠牲の上に成り立つ資源をリサイクルするこ とを目的とした最初の試験である。 私の研究では、廃牛肉と鮭を細菌汚染することな く、加水分解するための技術を示しており、 最終的には、他の産業で利用される可能 性を示した。

3

Catalogue

1. Introduction ... 5

2. Part 1: A detailed introduction of HPCD decontamination technology ... 9

2.1 Factors influencing the inactivation effect of HPCD ... 11

2.1.1 Microorganism species ... 11

2.1.2 Cell concentration ... 14

2.1.3 pH and water content ... 14

2.1.4 Physical state of carbon dioxide... 15

2.1.5 Treatment time, Pressure, and Temperature ... 15

2.1.6 Combination with other technologies and additives effects ... 16

2.2 Inactivation mechanism of HPCD ... 18

2.2.1 Mechanism of HPCD inactivation in vegetative cells ... 18

2.2.2 Mechanism of HPCD inactivation of spores ... 24

2.3 HPCD inactivation on some enzymes ... 25

2.4 The conclusion of part 1 ... 29

3 Part 3: waste meat was hydrolyzed by industrial protease under HPCD treatment conditions. . 30

3.1 Materials and methods ... 30

3.1.1 Pre-treatment of materials ... 30

3.1.2 High pressure carbon dioxide ... 31

3.1.3 Industrial proteases selection. ... 31

3.1.4 Hydrolysate collection and analysis ... 33

3.1.5 Application of hydrolysate ... 33

3.2 Results and discussion ... 34

3.2.1 High pressure carbon dioxide decontamination ... 34

3.2.2 Industrial proteases selection ... 36

3.2.3 Hydrolysate analysis ... 36

3.2.4 The application of hydrolysates ... 38

4

3.3 The conclusion of part 3 ... 38

4 Part 3: waste salmon was hydrolyzed by industrial protease under HPCD treatment condition 40 4.1 Materials and Methods ... 41

4.1.1 Preparation of salmon ... 41

4.1.2 High-pressure carbon dioxide treatment ... 41

4.1.3 DNA extraction and 16S rDNA gene amplification ... 43

4.1.4 Bacterial DNA quantification by real-time PCR and bacterial community analysis by NGS 43 4.1.5 Enzymatic hydrolysis ... 46

4.2 Results and Discussion ... 47

4.2.1 HPCD DE determined by colony counting ... 47

4.2.2 HPCD decontamination efficiency determined by real-time PCR and NGS ... 50

4.2.3 Effect of HPCD on hydrolysis ... 56

4.3 The conclusion of part 4 ... 59

5 Acknowledgement ... 60

6 References... 63

5 1. Introduction

The United Nations included the reduction of food loss and waste in its Sustainable Development Goals (SDGs), as a result of land degradation, overfishing, deterioration of the marine environment, and other natural resource challenges (SDGs, 2018). Many communities, groups, and institutions have made significant efforts to reduce food loss and waste. September 29 was designated as the International Day of Awareness of Food Loss and Waste by the United Nations in 2019, at the 74^th United Nations General Assembly. The Farm-to-Fork strategy, launched on May 20^th 2020, may also contribute to reductions in food loss and waste in attempting to meet its goal towards a ‘fair, healthy, and environmental-friendly food system’ (Mowlds, 2020). Advanced recycling technologies for food waste are also essential to meet the SDGs, specifically Goal 12, which covers consumption and production. In particular, there is a substantial need to develop relatively inexpensive

technologies that provide greater economic benefit.

Among these waste foods, over 52.6 million ton waste meat is produced globally every year, which can have a harmful impact on our environment (FAO, 2012). The 2011–2016 reports from the Food and Agriculture Organization of the United Nations stated that approximately 12.7% of fish (20.7 million tons per year) were used for non-food purposes (FAO, 2018). From the aspect of resources, waste meat waste fish have great potential to be recycled. Since meat contains abundant amino acids, salmon is widely consumed and contains a broad variety of proteins and lipids (Exler and Pehrsson, 2007), waste meat and fish can be used as an immense source of amino acids for other industries following enzymatic hydrolysis which would offer relatively high economic benefits for

6 other industries. For example, waste meat and salmon may be recycled as animal feed additives, providing a balanced nutrition and preventing inflammatory bowel disease in animals. It has been demonstrated that amino acids have the potential to maintain intestinal integrity in inflammatory bowel disease patients (Liu et al. 2017).

Therefore, finding an appropriate way of handing waste food has become a significant issue.

Salminen et al. (2002) have described several waste meat disposal methods such as landfilling, composting, incineration, rendering, animal food and anaerobic digestion. Among these methods, rendering presents an advantage of a higher market value compared to other methods (Salminen and Rintala, 2002; Charles and David, 2016). However, it is not considered eco-friendly due to its high temperature requirement (more than 100℃) (Leon et al., 2018). These methods can be performed at a low cost; however, we have a history of environmental pollution and bovine spongiform

encephalopathy.

The enzymatic hydrolysis of waste meat and salmon is a feasible means to decompose waste meat and salmon into smaller peptides and amino acids. However, bacterial contamination needs to be considered, particularly contamination by pathogenic bacteria as a result of the mild temperature used in enzymatic hydrolysis and the high bacterial counts of fish and fish products (Gram and Huss, 1996). As such, it is essential to decontaminate waste meat and salmon during enzymatic hydrolysis.

In the practical application of industrial enzymatic hydrolysis, addition of bacteriostatic or bactericidal preservatives (like ammonia fibre expansion) and application of high temperature are always used to prevent biomass from getting contaminated. This makes the industrial process

7 uneconomical, inconvenient and brings pressure to the environment (Pasupuleti and Braun, 2010;

Serate et al., 2015). The novel methods such as UV treatment, pulse electric field application are under investigation for preventing contamination (Uchida et al., 2008). In my case, as a novel decontamination technique, high-pressure carbon dioxide decontamination technology was applied in enzymatic hydrolysis process to prevent the biomass from getting contaminated by microbes. The hydrolysates from the waste meat and salmon can be used in microbial culture media in laboratory and can possibly be used as an additive for animal feed.

High-pressure carbon dioxide (HPCD) treatment is a novel non-thermal decontamination technology that inactivates microorganisms at mild temperatures. Many studies have demonstrated the inactivation of many bacterial spores using HPCD treatment (Thakur et al. 2013; Rao et al.

2015). The inactivation of microorganisms using HPCD has been attributed to cell membrane damage, which disrupts cell metabolism and damages cellular organelles (Takahashi et al. 2019;

Tamburini et al. 2014; Niu et al. 2017a; Yu et al. 2020). Low water content reduces the inactivation efficiency of HPCD (Chen et al. 2017); as such, HPCD is an unsuitable means to decontaminate the solid matrix. Despite this disadvantage, HPCD decontamination still offers many advantages including the relatively low cost and benign nature of CO2, mild treatment temperature, low treatment pressure (generally below 10 MPa), and relatively minimal equipment requirements.

HPCD-treated food has been proven safe for animals, indicating that no toxic compounds are produced during HPCD decontamination (Hibi et al. 2019). HPCD ensures food safety from microorganisms and removes specific chemicals; it has been shown to effectively extract specific undesired components. For example, 81 % of vicine was removed from faba beans following HPCD

8 treatment at only 40 °C and 4 min with 8 bar (Polanowska et al. 2019).

My doctoral thesis was composed by 3 parts:

Part 1: a detailed introduction of HPCD technology was given. To support the application of HPCD on decontaminating waste meat and salmon during enzymatic hydrolysis process, the existing information on high-pressure carbon dioxide decontamination technology was reviewed. HPCD bactericidal effects and its bactericidal mechanism was also discussed (Yu et al., 2020).

Part 2: waste meat was hydrolyzed by industrial protease under HPCD treatment condition. In this part, 50℃ under 1 MPa HPCD for 16 h could achieve an optimum decomposition efficiency and have no influence on industrial proteases activities during enzymatic hydrolysis process. Orientaaze OP from HBI Enzymes Inc. was selected as the best industrial protease (possessing the highest hydrolysis efficiency) from 10 different industrial protease. The hydrolysates recycled from beef meat under HPCD treatment condition can be applied for microbial culture mediums (Yu and Iwahashi, 2019).

Part 3: waste salmon was hydrolyzed by industrial protease under HPCD treatment condition. In this part, Real-time PCR analysis demonstrated that HPCD was able to inhibit an increase in bacteria at moderate temperatures. Based on NGS (Next Generation Sequencing) analysis, there was a very low abundance of Bacillus and some histamine producers, such as Pseudomonas, Acinetobacter, Enterobacter, and Klebsiella, detected in samples treated using HPCD at 50 ℃ and 1 MPa for 16 h.

Hydrolysate analysis showed that HPCD treatment at 1 MPa did not affect the hydrolysates from salmon. By applying real-time PCR and NGS, the safety of hydrolysates was ensured.

9 My works will support the application of HPCD in industrial enzymatic hydrolysis and increase the sustainability of bio-based materials.

2. Part 1: A detailed introduction of HPCD decontamination technology

HPCD is a type of non-thermal pasteurization that applies pressurized CO2, at between 0.1 MPa (1 bar) and 50 MPa (500 bar). Compared with high pressure hydrostatic pressure decontamination technology, HPCD uses less sophisticated equipment, since most microbes can be inactivated at under 50 MPa. HPCD possesses great potential as a novel, non-thermal decontamination technology, the bactericidal effects on various microorganisms and its bactericidal mechanism are the main subjects of this part.

Since Valley and Rettger, in 1927, discovered the bactericidal effect of pressurized CO2, an increasing number of scientists have conducted research on HPCD inactivation in microorganisms, as a novel nonthermal pasteurization technology for the food industry. In the year 2012, the number of published papers about HPCD inactivation on food increased notably (Figure 1). Although HPCD decontamination has yet to reach a large commercial scale, more people are realizing its potential as a technology, following significant, positive results from research concerning its bactericidal effect (Cheng et al. 2011). HPCD is a novel non-thermal decontamination technology that applies pressurized CO2 at ≥ 0.1 MPa (1 bar), at a relatively low temperature (lower than thermal pasteurization). Carbon dioxide presents different phases at different temperatures and pressures (Figure 2). Above critical conditions (7.38 MPa, 31.1 °C or 73.8 bar, 304.25 K), CO2 always exists as a supercritical fluid, which has properties of gas and liquid (Sapkal et al. 2010). Until now, HPCD

10 Figure 1. The yearly published papers in PubMed about High Pressure Carbon Dioxide on Food.

Figure 2. Schematic representation of Carbon Dioxide Phase Diagram.

0 50 100 150 200 250 300 350

1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018

Number of published papers

Yearly published papers in PubMed about HPCD on Food.

11 inactivation studies have generally been conducted at temperatures ranging from room temperature (25 ℃ or 298.15 K) to 100 ℃ (373.15 K) and pressures ranging from 0.1 MPa to 50MPa. Hence, for HPCD inactivation technology, carbon dioxide is generally found in the gas phase or as a

supercritical fluid (Figure 2.).

In this part, discussions of the applications of HPCD in inactivating bacteria, spores, and were presented. The lack of clarity and details regarding the mechanisms of HPCD inactivation of microorganisms are major obstacles to its industrial application. Therefore, possible mechanisms of bactericidal activity are fully discussed in this part, including information from recent studies. In this part, the issues of HPCD technology, faced by the food industry, and present some suggestions for future work are also discussed.

2.1 Factors influencing the inactivation effect of HPCD

The bactericidal effect of compressed CO2 has been known for nearly a hundred years (Valley and Rettger 1927). It has been shown that the efficiency of HPCD in microorganism inactivation is related to many factors. Since many of these factors influence pasteurization efficiency, to varying degrees, a discussion of HPCD inactivation factors should be presented here. We also expect this to provide useful guidance for industrial operation.

2.1.1 Microorganism species

The bactericidal effects of HPCD on various bacteria have been well studied by scientists (Table 1). Different bacteria present different responses to HPCD treatment. It was observed that aerobic psychrophilic microorganisms are much more sensitive to HPCD than aerobic mesophilic

12 Table 1: Inactivation of HPCD on microorganism. AMM (aerobic mesophilic microorganisms); APM (aerobic psychrophilic microorganisms); SPS (sterile physiological saline); CSF (coho salmon fillets); RCBM (raw chicken breast meat); RT (room temperature); HHP (high hydrostatic pressure).

Microorganism species Solution HPCD condition Additional technique Inactivation efficiency Reference

Escherichia coli Pineapple juice 10 MPa, 31 ℃, 3.06 min Ultrasound Total inactivation Paniagua-Martínez et al. 2018

Wet E. coli AW1.7 LB broth 35 ℃, 15 min, 10 MPa No More than 3 log (CFU/ml) reduction Chen et al. 2017

Dry E. coli AW1.7 Dry 35 ℃, 15 min, 10 MPa No Less than 0.5 log (CFU/ml) reduction Chen et al. 2017

Yeast Pineapple juice 10 MPa, 31 ℃, 3.06 min Ultrasound Total inactivation Paniagua-Martínez et al. 2018

Yeasts and moulds Strawberry juice 60 MPa, 40 ℃, 30 min No Total inactivation Marszalek et al. 2015

Vibrio parahaemolyticus SPS 25 MPa, 40 ℃, 10 min No 5.69 [ − Log(N/N0)] Xu et al. 2017

Vibrio parahaemolyticus SPS 25 MPa 40 ℃, 40 min No 7.02 [ − Log(N/N0)] Xu et al. 2017

Saccharomyces pastorianus SPS 43 ℃, 1 MPa, 1 min; 37 ℃, 2 MPa, 1 min 5% ethanol addition 6 log reduction (both) Kobayashi et al. 2020

AMM CSF RT, 5 min, 150 MPa+70 % CO2 HHP 0.43 log reduction Perez-Won et al. 2020

APM CSF RT, 5min, 150 MPa+70 % CO2 HHP More than 0.67 log reduction Perez-Won et al. 2020

Mesophilic viable bacteria Pineapple juice 10 MPa, 31 ℃, 3.06 min Ultrasound Total inactivation Paniagua-Martínez et al. 2018

Mesophiles RCBM 14 MPa, 40 ℃, 45 min No 2.99 Log CFU/g reductions González-Alonso et al. 2019

Yeasts and molds RCBM 14 MPa, 40 ℃, 45 min No 4.01 Log CFU/g reductions González-Alonso et al. 2019

13 microorganisms (Perez-Won et al. 2020). Furthermore, L. monocytogenes is much more sensitive than E. coli to HPCD treatment (Fleury et al. 2018).

However, no pattern of bactericidal effect was found between gram-positive bacteria, gram- negative bacteria, or fungi, as far as current research goes. This suggests that the bactericidal effects of HPCD have no significant connection with the cell wall. Nevertheless, in the interest of scientific understanding, more research needs to be conducted to clarify the bactericidal effects of HPCD on gram-positive and gram-negative bacteria, and on fungi, by varying and controlling the treatment conditions (temperature, pressure, time, and medium).

Although bacterial vegetative cells can be easily inactivated by HPCD, their spore forms are more difficult to inactivate by HPCD, in the same conditions of pressure, time, and temperature. For example, a relatively high temperature (∼85 °C) combined with 20 MPa HPCD for 60 min is needed, to effectively inactivate spores of Bacillus subtilis (Rao et al. 2019). However, more than 7 log reduction of vegetative cells of the same species can be completed by treatment at 38 ℃ and 7.4 MPa for just 2.5 min (Spilimbergo et al. 2002). The resistance of spores to HPCD has been ranked as follows: B. subtilis > G. stearothermophilus > B. licheniformis > B. coagulans > B. cereus (Rao et al.

2015).

It was also discovered that HPCD can inactivate bacteriophage T4 (Cheng et al. 2011). About a 4.0 log reduction for bacteriophage T4, > 3.0 log reduction for bacteriophage MS2, > 3.3 log reduction in bacteriophage Qβ and just under a 3.0 log reduction in bacteriophage ΦX174 were all achieved at 0.7 MPa for 25 min at 22 °C (Vo et al. 2013; Vo et al. 2014).

14 2.1.2 Cell concentration

Bacterial samples with a higher concentration of cells show a lower efficiency of inactivation by compressed carbon dioxide than low-concentration samples (Fleury et al. 2018). When higher concentrations were treated with pressurized CO2, more cells clumped, which made it difficult for CO2 to penetrate the cell membranes, leading to a decreased bactericidal effect. Consequently, in real industrial applications, the initial concentration of cells should be taken into consideration for the pasteurization of biomass by HPCD.

2.1.3 pH and water content

Some authors found that the efficiency of microbial inactivation by HPCD could be enhanced by decreasing the initial environmental pH (Chen et al. 2010). However, regardless of the treatment pressure, the strain of microorganism, or exposure time, the pH of the endpoint after carbon dioxide treatment showed no significant difference (Fleury et al. 2018). The effect of pH was also

investigated when the bacteriophage virus MS2 was treated with HCl (hydrochloric acid) under the same pressure as used for HPCD, and CO2 treatment presented a higher sterilization effect than HCl treatment (Vo et al. 2013). Hence, the pH decrease, which is induced by HPCD, is not the reason for microbial inactivation, but a lower initial environment pH can still improve the efficiency of inactivation.

Water has an important role in the inactivation efficiency of HPCD. A higher water content could enhance bactericidal efficiency. It has been suggested that wet cells are more sensitive to HPCD than dry cells (Chen et al. 2017). For example, when wet E. coli in lysogeny broth was treated at 35 °C at

15 10 MPa with HPCD, for 15 min, cell colonies were reduced by more than 3 log (CFU/mL).

However, when the dry E. coli was placed in the same conditions, cell colonies were reduced by <

0.5 log (CFU/mL). Other researchers have also demonstrated that water content plays an essential role in the bactericidal effect of HPCD (Vo et al. 2015). Generally, HPCD technology presents a better inactivation efficiency for liquid materials compared to solids.

2.1.4 Physical state of carbon dioxide

One interesting study showed that gaseous CO2 presents higher inactivation efficiency than supercritical CO2 or liquid CO2. In particular, when dry E. coli was treated with HPCD, more bactericidal activity can be accomplished with gaseous CO2, whereas dry E.coli are resistant to treatment by supercritical and liquid CO2 at the range of temperatures used (Chen et al. 2017).

However, a slightly higher temperature (~ 65 ^oC) is still required for effective inactivation of dry E.

coli.

2.1.5 Treatment time, Pressure, and Temperature

Generally, CO2 treatment had an enhanced efficiency in cell destruction when the duration of exposure was increased. For example, 1.4 log CFU/g reduction of E. coli can be achieved after 15 min of treatment, whereas a 45 min treatment can induce up to 5 log reduction (González-Alonso et al. 2019). However, longer treatment periods are not always worth the effort to increase inactivation.

Fleury et al. (2018) found that the efficiency increased with time, until the treatment period reached a certain point, and there was no further improvement at 50 ℃, even when the treatment was prolonged.

16 A higher inactivating effect of CO2 could be achieved by using higher pressure (Marszalek et al.

2015; Chen et al. 2017). Higher pressure can enhance CO2 solubilization, facilitating its contact with and penetration into cells, which could explain the higher inactivation efficiency. Higher pressure can generate the same inactivation efficiency, at lower temperatures, as lower pressure can at a higher temperature (Kobayashi et al. 2020). Pressure has a positive effect on HPCD inactivation efficiency, but is not as important as temperature (Fleury et al. 2018).

Among all the factors, temperature is considered to be the most important, to inactivate bacteria.

However, temperature is not a factor to be regarded independently for bacterial inactivation. Similar pictures of cell cytoplasmic damage were observed at 38 ℃and 50 ℃, but only the higher

temperature could achieve complete inactivation (Fleury et al. 2018). Despite this evidence, other studies suspect that pressure is the main lethal factor. According to them, a higher pressure can induce a decrease in pH, which leads to microbial inactivation (Marszalek et al. 2015).

Garcia-Gonzalez et al. (2007) described the schematic survival curves of bacteria with HPCD treatment. They explained that, during the HPCD process, the shapes of survival curves are related to the number of experimental data, the treatment time, and the pressure applied. Generally, sufficient experimental data tend to present a concave curve. However, fewer data are prone to show a log- linear curve (Figure 3. A and B), but when the pressure is increased, the shape of the curve is converted to concave (Figure 3. A).

2.1.6 Combination with other technologies and additives effects

Other factors also have some influence on the bactericidal effect of HPCD, such as agitation, the

17 Figure 3. Schematic representation of survival curve shapes (Vegetative microbial cells) during HPCD inactivation (a so-called shoulder period was removed out of discussion in this paper). A:

survival efficiency following time; B: survival efficiency following pressure.

18 physicochemical environment, and some additives. Appropriate agitation can enhance the

bactericidal efficiency of HPCD (Tsuji et al. 2005). The microbial inactivation rate could be enhanced by certain additives, such as ethanol (Kobayashi et al. 2020). Especially for spores, additives can greatly increase the inactivation effect at relatively mild temperatures (35–60 ºC) (White et al. 2006; Hemmer et al. 2006; Checinska et al. 2011). When ultrasound is combined with HPCD, total inactivation can be reached in just 3 min, under 10 MPa, at 31 °C (Paniagua-Martínez et al. 2018). Another study, using HHP with HPCD, showed that the bactericidal effect is mainly due to the CO2 content, and not the pressure, since a pressure of 150 MPa alone had no bactericidal effect, whereas CO2 alone could inactive the bacteria without increased pressure (Perez-Won et al.

2020). It has also been suggested that using a pressure < 200 MPa for HHP is not effective in extending the storage period for salmon (Briones et al. 2010). This suggests that pressure alone (at <

200 MPa) has a low efficiency of inactivation, without CO2.

2.2 Inactivation mechanism of HPCD

2.2.1 Mechanism of HPCD inactivation in vegetative cells

Many scientists have tried to explain the inactivation mechanism of HPCD. Although the details are still unknown, some theories have been more widely accepted, and will be discussed here with reference to published work, including some new findings. Additionally, we provide a novel direction for future studies, to clarify and improve the mechanisms of HPCD inactivation. A schematic diagram of mechanisms for the inactivation of vegetative microbial cells is shown in Figure 4.

19 Figure 4. Diagram of HPCD inactivation mechanisms on vegetative microbial cells.

20 A detailed interpretation is given, below, in points ○5 and ○6, since CO2-induced membrane alternation is considered as the main reason for microbial inactivation.

① When bacterial cells are subjected to pressure, cell hydrophobicity will increase which can make the cells clump more easily. At higher cell concentrations, more cells clump, which leads to a decrease of inactivation rate by HPCD (Fleury et al. 2018).

② Although cells are prone to clump under pressure, more severe shear force could be obtained from applying HPCD to separate cells. Therefore, higher pressure leads to a higher inactivation rate (Marszalek et al. 2015).

③ Pressured CO2 can cause cell surface damage and disruption to intracellular organization.

Under scanning and transmission electron microscopes (SEM and TEM), the alteration of microbial cell morphology can be visualized, after HPCD treatment. These alterations can be enhanced by longer treatment times (Liao et al. 2010; Niu et al. 2017a). A large number of bulges were found on the extracellular surface of HPCD-treated cells, which indicates the leakage of cytoplasmic contents. However, morphological alteration is not necessarily the reason for cell death, because inactivated cells can still seem to be integral, under the TEM (Fleury et al. 2018).

④ CO2 diffusivity is enhanced at high pressure and extracellular pH is decreased. Dissolved CO2

can convert to HCO3- and CO32-. Meanwhile, H⁺ can be released from H2O. In the aqueous extracellular environment, the pH will decrease due to release of H⁺, which can also increase membrane permeabilization, to allow more CO2 to enter the cytoplasm. This explains why the

21 HPCD inactivation efficiency of dry strains was greatly decreased. However, regardless of the liquid matrix, the pH induced by the pressured CO2 will not decrease constantly with pressure and temperature.

⑤ Membrane permeabilization and fluidity are enhanced.

About 81 % of cells were permeabilized and 18 % of cells were partially permeabilized after 15 min of CO2 treatment under pressure (12 MPa and 35 °C). Additionally, the ratio of phosphoglyceride to phosphatidylethanolamine decreased, which indicated that the stability of the E. coli membrane also decreased under approximately 12 MPa of pressured CO2 treatment (Tamburini et al. 2014). This study confirmed the increase in cell membrane permeability and the leakage of intracellular cytoplasmic solutes which may be essential for microbial growth.

Another research showed that HPCD can lead to a decrease in total saturated fatty acid content and an increase in total unsaturated fatty acid content (Xu et al. 2017). Subsequently, the stability and permeabilization of cell membranes are altered. Membrane damage in a large majority of cells was also observed in the work of Fleury et al. (2018). Kobayashi et al. (2020) also found that higher temperatures can increase the phase transition of the membrane, which may disrupt cytoplasmic membrane permeabilization.

Another study also found that the disruption of the endoplasmic reticulum, nuclear membrane, Golgi body, and nucleolus caused a reduction in yeast cell viability when Saccharomyces cerevisiae was treated by HPCD. The membranes of major organelles were damaged by HPCD treatment, rather than the cell membrane (Takahashi et al. 2019). Their investigation proved that

22 membrane damage is also a lethal cause for eukaryotes.

⑥ Decreased pH and HCO3- (produced from CO2) in the membrane can destroy the membrane surface charge balance and alter membrane functions.

The lower pH produced by pressured CO2 may decrease the activity of some membrane proteins, and become deleterious to their biological functions. HCO3− ions produced by pressured CO2 can change protein–lipid electrostatic interactions. For example, the topology of the inner membrane protein is believed to be influenced by the presence of negatively charged phospholipids (Xie et al. 2006), which may be changed by HCO3− ions. The localization of the charged amino acids in the membrane may also be influenced by HCO3− ions. It should be noted that the conformation and function of membrane proteins are related to the composition and distribution of membrane lipids (Ma et al. 2017). Hence, alteration of membrane protein activity may influence the composition and distribution of membrane lipids. This explanation was corroborated by a study of lipid A, which plays an important role in cell survival and is

distributed in the periplasmic space (Xu et al. 2017). However, more studies are needed on how HCO3− ions and decreased pH might influence the functioning of membrane proteins.

Furthermore, there are many DNA-specific attachment sites on cell membranes. These are required for DNA replication and for the process of cell segmentation (Parks et al. 1982). As we have accepted that cell membranes can be altered significantly by HPCD, the possible alteration of DNA-specific attachment sites should be taken into consideration. This might provide more evidence for the mechanisms of the bactericidal effect of HPCD, and is another avenue for

23 future research.

⑦ Some proteins and enzymes may lose their activities, due to HPCD treatment. The activity of Na+/K+-ATPase, the main enzyme that maintains the balance of various substances inside and outside of cells, has been shown to be significantly decreased by HPCD, especially under higher pressure and longer treatment times (Xu et al. 2017). Alkaline phosphatase, a periplasmic enzyme, can also be deactivated at lower temperatures with HPCD than with thermal treatment alone (Kobayashi et al. 2020). However, the intracellular pH change brought about by pressured CO2 is not a major contributor to lethality (Chen et al. 2017). Decreased pH was considered as the reason for some protein deactivation. However, the latest research suggests that protein deactivation by HPCD is due to an ‘interfacial denaturation’ mechanism. In their recent study, Monhemi and Dolatabadi (2020) used a molecular dynamics simulation method to clarify the mechanism of HPCD inactivation of proteins and enzymes. They suggested that protein denaturation occurs at the CO2/water interface in a HPCD pasteurization system. They found that hydrophobic regions in the protein cores were expanded upon arrival at the CO2/water interface. First, when HPCD starts, proteins and enzymes become accessible to the CO2/water interface. Pressurized micro-size CO2 bubbles and mixing in the processing vessel can both promote this movement. Second, hydrophobic protein regions start to unfold, from globular to flat and extending, in conformation. Third, tertiary protein structure undergoes change, followed by protein denaturation. This may also cause functional alterations in the membrane.

⑧ Internal ribosomes and CO32- (produced from CO2) induce intracellular precipitation. Anions

24 such as CO32- or HCO3- , may precipitate intracellular inorganic electrolytes (Ca²⁺), leading the cytoplasmic interior to lose its electrolytic balance. Subsequently, with increased permeability, these inorganic electrolytes and other constituents may transfer to the extracellular environment through the cell membrane. This has been confirmed by some studies. After HPCD treatment, an obvious increase was found in the types of protein in the supernatant, which indicated that the constituents of the cells were transferred to the extracellular space (Xu et al. 2017). When intracellular constituents are removed, the structure of the bio-membrane and the balance of the biological system may be altered, which could enhance the lethal effect of pressurized CO2.

In addition, internal ribosomes and intracellular materials can be agglomerated or precipitated, followed by uneven distribution at the cell membrane (Xu et al. 2017). Precipitation of

intracellular ribosomes can, subsequently, cause confusion in gene expression.

⑨ The metabolic pathways that require CO2 as a reagent can be stimulated. Those that can produce CO2 will be inhibited. Phosphatidylcholine synthesis increases with pressurized CO2 treatment.

This can explain the enhanced stability of some cell membranes and cellular resistance to HPCD inactivation (Niu et al. 2017a), as bacteria present enhanced adaptability to the adverse external environment. The metabolism of the urea cycle is also significantly enhanced, along with the induction of urea cycle-related genes (Niu et al. 2017b). These studies revealed that HPCD can influence cell metabolism.

2.2.2 Mechanism of HPCD inactivation of spores

Spores usually possess a high ability to resist physical and chemical treatment, due to their unique

25 structure (Setlow 2006). Spore structure includes an exosporium, a coat layer, outer membrane, cortex, germ cell wall, inner membrane, and core (Leggett et al. 2012) (Figure 5). The permeability barrier is composed of an exosporium, a coat layer, and a cortex that contributes to spore resistance against pressure or attack by chemicals and lytic enzymes (Setlow 2006).

Generally, spore inactivation by HPCD proceeds as follows. When heat is applied to spores, the enzymes in the spores may be activated, which may lead to cell modification. This provides an opportunity for CO2 to penetrate the cell, followed by damage to the cell structure and metabolic systems. Subsequently, the fluidity and permeability of the inner membrane are increased by the HPCD. The evidence shows that HPCD treatment can trigger the loss of core materials from spores, such as metal ions and dipicolinic acid (DPA) from the inner membrane (Rao et al. 2016; Rao et al.

2020). Due to an increase in the permeability of the inner membrane, the heat resistance of the spore is also reduced by HPCD treatment. It should be noted that germination is not the reason that heat resistance of spores is reduced, as HPCD-treated spores did not undergo typical germination due to the damage of germinant receptors. Then, spore outgrowth is blocked due to the absence of germination, which leads to spore death (Rao et al. 2019; 2020).

2.3 HPCD inactivation on some enzymes

HPCD can promote the inactivation of enzymes. Especially in some fruits, vegetables, and some related products, peroxidase and polyphenol oxidase are the most important enzymes to negatively affect food quality by browning, the formation of off-flavors, and the loss of vitamins and pigments. The inactivation of peroxidase and polyphenol oxidase activity by HPCD is dependent on

26 Figure 5. Diagram of spore inactivation by HPCD.

27 temperature, pressure, and time (Table 2). The remaining peroxidase activity was 61.39 % after treatment with CO2 at 20 MPa and 45 ℃ for 20 min, whereas, only 29.32 % peroxidase activity was preserved when the temperature increased to 65 ℃. Without HPCD, peroxidase activity remained at 94.56 %, even when the temperature was increased to 65 ℃ (Murtaza et al. 2020). When the CO2

pressure increased to 30 MPa for 30 min, 83 % of the peroxidase was effectively inactivated (Marszalek et al. 2015). Compared with peroxidase, polyphenol oxidase is much more sensitive to HPCD treatment. HPCD conditions of 30 MPa and 45 ℃ for 30 min can totally inactivate

polyphenol oxidase (Marszalek et al. 2015).

The CO2 concentration is more important than the pressure, for inactivating enzymes. When 70 % CO2 was applied without pressure, 51 % of protease was inactivated. When 150 MPa was applied, without CO2, the inactivation was only 20 %. The same results were obtained for collagenase activity. When 150 MPa pressure was applied with 100% CO2, the inactivation of collagenase did not increase compared with 0 MPa pressure and 100 % CO2 treated collagenase (Table 2) (Perez- Won et al. 2020). Beside these examples, many other enzymes have been successfully inactivated by HPCD (Benito-Román Ó et al. 2019b; Iftikhar et al. 2014; Kobayashi et al. 2016; Liao et al. 2009;

Marszałek et al. 2019; Manzocco et al. 2017; Ceni et al. 2016).

For a possible mechanism by which HPCD might inactivate enzymes, Benito-Román et al.

(2019a) found, by fluorescence spectroscopy analysis, that HPCD can trigger significant changes in an enzyme's tertiary structure. Following this result, as previously mentioned, Monhemi and

Dolatabadi (2020) found that the tertiary structure of proteins and enzymes is significantly changed

28 Table 2: Inactivation of HPCD on enzymes. CSF (coho salmon fillets); RT (room temperature); HHP (high hydrostatic pressure).

Enzymes Food species Conditions Additional technique Results Reference

Peroxidase Strawberry juice (30, 60 MPa), 45 ℃, 30 min No 83 %, 88 % inactivated Marszalek et al. 2015

Peroxidase Apple juice 20min, 65 ℃ No 5.44 % inactivated Murtaza et al. 2020

Peroxidase Apple juice 20 MPa, 20 min, 45 ℃ No 38.61 % inactivated Murtaza et al. 2020

Peroxidase Apple juice 20 MPa, 20 min, 65 ℃ No 70.68 % inactivated Murtaza et al. 2020

Polyphenol oxidase Strawberry juice (30, 60 MPa), 45 ℃, 30 min No 100 % inactivated (both) Marszalek et al. 2015

Polyphenol oxidase Apple juice 20 MPa, 20 min, 45 ℃ No 81.55 % inactivated Murtaza et al. 2020

Polyphenol oxidase Apple juice 20 MPa, 20 min, 55 ℃ No 100 % inactivated Murtaza et al. 2020

Protease CSF RT, 5 min, 0 MPa + 70 % CO2 HHP 51 % inactivated Perez-Won et al. 2020

Protease CSF RT, 5 min, 150 MPa + 50 % CO2 HHP 20 % inactivated Perez-Won et al. 2020

Protease CSF RT, 5 min, 150 MPa+100 % CO2 HHP 59 % inactivated Perez-Won et al. 2020

Collagenase CSF RT, 5 min, 0 MPa + 100 % CO2,

10 days storage HHP 91 % inactivated (compare

with 0 MPa and 0 % CO2) Perez-Won et al. 2020 Collagenase CSF RT, 5 min, 150 MPa + 100 %

CO2, 10 days storage HHP 91 % inactivated (compare

with 0 MPa and 0 % CO2) Perez-Won et al. 2020

29 from globular to flat and extended conformations. The alteration of protein structure is considered to be the main cause of inactivation.

2.4 The conclusion of part 1

It has been confirmed that HPCD could be applied as a novel, nonthermal decontamination technology, since the bactericidal effect of HPCD has been widely tested on various microorganisms.

However, a lack of ample studies prevents HPCD technology from growing on an industrial scale.

Although the inactivation of microorganisms by HPCD has been studied extensively, the detailed mechanisms are still unclear. Until now, studies have shown that the alteration of the cell membrane is the leading cause of bactericidal activity. Hence, in future works, scientists should give more consideration to membrane alteration by HPCD treatment, whose possible mechanisms we have introduced at length. To date, studies on the inactivation of spores and viruses by HPCD have been insufficient, especially on some food pathogens, such as Clostridium spores, which can cause spoilage, and foodborne illness. In addition, a further study of the mathematical modeling of the inactivation kinetics of various microorganisms should be conducted to provide data for industrial application.

Owing to the lower requirement for pressure (lower than 50 MPa) compared with high pressure hydrostatic technology, HPCD has great advantages, such as low cost in equipment, larger scale production, and is much safer for operators. In addition, CO2 is cheap and is considered “green,” in the context of food science, without toxicity. However, the process parameters (pressure,

temperature, and treatment time) of HPCD for different matrix need to be studied energetically.

30 More efforts are needed to provide more convincing data for future, commercial-scale applications.

3 Part 3: waste meat was hydrolyzed by industrial protease under HPCD treatment conditions.

In this part, minced beef meat was hydrolyzed by 10 different industrial proteases under HPCD treatment condition. According to this work, 50 ℃ under 1 MPa HPCD for 16 h could achieve an optimum decomposition efficiency and have no influence on industrial proteases activities during enzymatic hydrolysis process. Orientaaze OP from HBI Enzymes Inc. was selected as the best industrial protease (possessing the highest hydrolysis efficiency) from 10 different industrial protease.

The hydrolysates recycled from beef meat under HPCD treatment condition can be applied for microbial culture mediums (Yu and Iwahashi, 2019).

3.1 Materials and methods

3.1.1 Pre-treatment of materials

Minced beef meat (MBM) was bought from Kanou meat shop of Gihei Corporation, Japan. Every 100 g MBM was mixed with 100 ml pure water for HPCD treatment. After mixing MBM with pure water, the connective tissue was removed from beef meat proteins by stainless steel mesh filter to obtain crude beef protein (CBP) solution. Subsequently, CBP solution was centrifuged (1594 g, 20 min, 4℃, TOMY CAX-371) to remove supernatant (excess water). The obtained CBP (the sediment after centrifugation) was further used for IP treatment.

31 3.1.2 High pressure carbon dioxide

In this study, HPCD was applied to prevent MBM from getting contaminated by microbes. Every tube containing 20 ml pre-treated MBM was transferred into a high-pressure vessel (30-11HF4, High Pressure Equipment, Elie, PA, USA, volume 794 cm³), which was placed in a thermostatic bath to control temperature. According to Japan’s High Pressure Gas Safety Act, applied pressure in our research was controlled below 1 MPa. Treatment duration was adjusted at 16 h as it was appropriate for industry workers’ work-rest schedule. Enzymatic hydrolysis process was conducted simultaneously with HPCD treatment. The standard culture medium (0.25% yeast extract, 0.5%

polypeptone, 0.1% D(+)-glucose, 1.5% agar powder, sterilized by autoclaving) was used to check the efficiency of decontamination. Plates which were spread with HPCD-treated samples were cultured in 30℃ incubator for 3 days. Colonies were observed subsequently.

3.1.3 Industrial proteases selection.

Ten different industrial proteases (IP) from several companies were tested to find the IP with the highest hydrolysis efficiency. Detailed information of ten different IP is concluded in Table 3. Gram- based method was used as IP addition method, since other enzymes and some additives in IP may influence the IP activity. 100 μl IP solution (0.05 g/ml) of ten different IP was added into 10 ml CBP slurry sample separately (CBP slurry contained 1 g CBP mixed with 1 ml pure water). Soluble protein concentration was measured by TaKaRa Bradford Protein Assay Kit (Takara Bio Inc.) and read by spectrophotometer (AS ONE, ASV11D, 595 nm). L-Glutamic Acid concentration was measured by L-Glutamic Acid kit (R-Biopharm AG, Art. No.:10139092035) and read by spectrophotometer (AS

32 Table 3. 10 different industrial protease information.

Name No. Company

Activity unit (u/mg)

Protease content

Appropriate temperature

(℃)

Appropriate pH

Buromerain F 1 A 800 + / ~60 2.0~10.0

Purotin SD-NY10 2 A 70 + / ~50 5.0~7.0

Papain W-40 3 A 400 + / ~80 3.0~9.0

Puroteaaze YP-SS 4 Y 80 + / 40~60 2.0~5.0

Magunakkusu MT-

103 5 R / / / /

Entiron NBS-100 6 R 83 + / ~50 5.0~7.0

Orientaaze AY 7 H / 70% / /

Orientaaze OP 8 H / 50% / /

Orientaaze 22BF 9 H / 28% / /

Nukureisin 10 H / 3% / /

A—Amano Enzyme Japan, Y— Yakult Pharmaceutical Industry Co., Ltd, R— Rakuto Kasei Industry Co., Ltd, H— HBI Enzymes Inc. [/]—No information available.

33 ONE, ASV11D, 492 nm) following 3K pore size filtration.

3.1.4 Hydrolysate collection and analysis

1 g of selected IP powder and 50 ml of pure water were mixed well with every 100 g of MBM.

After enzymatic hydrolysis under HPCD treatment, samples for component analysis were heated at 80℃ in a water bath (THEMO MAX TM-2, AS ONE) for about 20 min to stop the enzymatic reaction followed by centrifugation (1594 g, 20 min, 4℃, TOMY CAX-371). The supernatant was subsequently used for serial filtrations (3 different pore size filtrations: 5.0 µm, 0.2 µm, 10 K).

Subsequently, the hydrolysate solution from MBM was collected.

This step was conducted thrice under the same conditions and the hydrolysate solutions were mixed together for analysing free amino acids and total nitrogen concentration.

Hydrolysate analysis was performed by Food Analysis Technology Center, SUNATEC, Japan.

According to this company, high-performance liquid chromatography (HPLC) was used to analyze free amino acids concentration (without prior acid hydrolysis) and Kjeldahl method was applied to test total nitrogen concentration.

3.1.5 Application of hydrolysate

Two novel types of culture media were prepared by our hydrolysate based on same nitrogen content compared with commercial culture media. Lysogeny broth (LB) for Escherichia coli K12JM109 cultivation: Commercial LB is composed of 1% BD Bacto™ tryptone, 0.5% BD Bacto™ yeast extract and 1% sodium chloride. Carbon-beeftone LB was composed of 8.15% HPCD-hydrolysate solution and 1% sodium chloride. Control-beeftone LB was composed of 8.15% Control-hydrolysate solution

34 and 1% sodium chloride. Yeast Extract Peptone Dextrose (YPD) for Saccharomyces cerevisiae S288C growth: Commercial YPD is composed of 2% BD Bacto™ tryptone, 1% BD Bacto™ yeast extract and 2% sodium chloride. Carbon-beeftone YPD was composed of 16.3% HPCD-hydrolysate solution and 2% sodium chloride. Control-beeftone YPD was composed of 16.3% Control-hydrolysate solution and 2% sodium chloride. After preparing the culture media, they were sterilized by autoclaving. The growth curves of E. coli K12JM109 and S. cerevisiae S288C were analyzed in a 96-well plate and read by microplate reader (TECAN Wako) at 600 nm.

All experiments were conducted at least three times.

3.2 Results and discussion

3.2.1 High pressure carbon dioxide decontamination

Some researchers have proved that higher temperature could enhance bacterial inactivation efficiency under HPCD (Spilimbergo and Bertucco, 2003; Erkmen, 2000). Three different

temperatures were tested to inactive MBM microorganisms under 1 MPa HPCD for 16 h. As shown in Figure 6, after 16 h treatment, large number of colonies grown in plates A, B, D, E. In plate C, less than 30 colonies were observed, whereas plate F did not show any colonies. Microbial growth decreased to some extent by HPCD treatment at the temperatures 40℃ and 45℃. However, large number of microorganisms still survived in plates D and E. Even though plate C showed significant decrease in microbial growth, contamination threat still existed. Only in plate F (50℃, 1 MPa HPCD, 16 h), microbial contamination of MBM could be completely prevented. This could provide a novel method to prevent biomass from getting contaminated by microorganisms during industrial

35 Figure 6. The effect of temperature and HPCD on meat bacterial survival. The treatment condition:

A, 40℃ 0.1 MPa without HPCD; B, 45℃ 0.1 MPa without HPCD; C, 50℃ 0.1 MPa without HPCD; D, 40℃ 1 MPa with HPCD; E, 45℃ 1 MPa with HPCD; F, 50℃ 1 MPa with HPCD.

A B C

D E F

36 enzymatic hydrolysis process.

3.2.2 Industrial proteases selection

Ten different IP were tested to select the IP with the highest hydrolysis efficiency. It is expected that the IP which showed higher hydrolysis efficiency should release more free amino acids, resulting in less sediment and less residual soluble protein. When the MBM was hydrolysed by acid hydrolysis, glutamic acid was found to be the most abundant amino acid in beef (Samicho et al., 2013). Hence, in IP selection experiment, L-Glutamic Acid (L form is widely occurring in nature) concentration was measured as an indicator of free amino acid content. According to Figure 7, No. 8 IP could produce the highest sediment reduction rate, the highest L-Glutamic Acid concentration and the lowest soluble protein concentration after the treatment. Despite the differences in protein concentration and activity unit of these IP, Orientaaze OP was selected as the best IP in this study, since only Orientaaze OP fulfilled our purpose that the more free amino acids the better.

3.2.3 Hydrolysate analysis

1％ Orientaaze OP IP addition for MBM hydrolysate manufacturing was a huge amount.

However, we found that 0.5% addition of Orientaaze OP IP produced less free glutamic acid (an indicator for hydrolysates), whereas 2% addition produced almost the same amount of free glutamic acid as observed with 1% addition, according to our current experimental conditions (data not shown). Free amino acids contents of HPCD treatment and non-HPCD treatment were measured to find whether HPCD has any influence on the hydrolysis process. As shown in Figure 8, none of the free amino acids showed a significant difference in concentration between HPCD treatment

37 Figure 7. Industrial Protease selection. A, Sediment reduction rate, Wet weight reduction/wet weight before treatment； B, L-Glutamic Acid concentration after treatment (g/L)； C, The reciprocal of soluble protein concentration (μg/ml) after treatment.

Figure 8. The influence of HPCD on hydrolysis process. HPCD Treat is the group which is treated under 1 MPa 50℃ HPCD for 16 hours. Control is the group which is treated under 50℃ 0.1 MPa without HPCD in a sealed tube for 16 hours.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 1 2 3 4 5 6 7 8 9 10

A

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 1 2 3 4 5 6 7 8 9 10

g/L

B

0 0.5 1 1.5 2 2.5 3 3.5

0 1 2 3 4 5 6 7 8 9 10

X10-3

C

0 200 400 600 800 1000 1200

Free Amino Acids content(mg) (every 100g hydrolysate)

HPCD Treat Control

38 hydrolysate and non-HPCD treatment hydrolysate. It means 1 MPa HPCD under 50℃ for 16 h has no influence on industrial enzymatic hydrolysis process.

3.2.4 The application of hydrolysates

LB culture medium and YPD culture media were prepared using our hydrolysates based on same total nitrogen content compared with commercial culture media developed for culturing E. coli K12JM109 and S. cerevisiae S288C, respectively. As shown in Figure 9, E. coli K12JM109 and S.

cerevisiae S288C showed excellent growth in our novel culture media, which were prepared using

MBM hydrolysates. Besides that, the growth curves of these two species showed no significant difference between our novel culture media and commercial culture media. Hence, we suggest that the hydrolysates produced as a result of MBM enzymatic hydrolysis along with our treatment (1 MPa HPCD 50℃ for 16 h) could be recycled for culturing microorganisms in laboratory.

3.3 The conclusion of part 3

According to our research, 50℃ under 1 MPa HPCD for 16 h could achieve an optimum

decomposition efficiency for minced beef meat. Orientaaze OP from HBI Enzymes Inc. was selected as the best IP in this work. 1% Orientaaze OP IP addition was used for MBM hydrolysate

manufacturing. We observed that none of the free amino acids showed a significant difference in concentration between HPCD treatment hydrolysate and non-HPCD treatment hydrolysate. It implies that HPCD technology could be applied in industrial enzymatic hydrolysis process for biomass decomposition. This provides a novel technology to be coupled with industrial enzymatic hydrolysis process to prevent the biomass from getting contaminated by microbes at relatively lower

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40 temperature. At last, the hydrolysates obtained by our methods from minced beef meat were used for culture media synthesis. E. coli K12JM109 and S. cerevisiae S288C were cultured in our novel culture media. Based on our results, the hydrolysate produced by our methods could be applied in the cultivation of microorganisms. Further applications have to be developed in order to make recycling of meat acceptable to the industry.

This report is the first trial aimed at recycling life-based resources, such as waste meat by using enzymatic reactions under HPCD. Further studies are required to optimize the amount of enzymes, and to elucidate the application and safety of recycled products.

4 Part 3: waste salmon was hydrolyzed by industrial protease under HPCD treatment condition

This study used industrial proteases to hydrolyze salmon meat into smaller compounds (amino acids) under HPCD conditions in an attempt to increase the sustainability of bio-based materials.

Next generation sequencing (NGS) and real-time PCR were applied to accurately determine the efficiency of HPCD decontamination in removing bacteria in salmon. We attempt to demonstrate a method to obtain safe hydrolysates from salmon as an industrial resource; it is anticipated that this method will serve as an advanced recycling technology supporting the non-food utility of fish. The outcomes of this study are also considered to increase the existing knowledge on HPCD

decontamination technology and promote the industrial application of HPCD.

41 4.1 Materials and Methods

4.1.1 Preparation of salmon

Salmon filets were purchased from Kanesue Gidaimaeten, Gifu City, Japan, and all skin was removed from the filets. Salmon (100 g) was mixed with 100 mL of pure water in a mixer and the mixed salmon slurry (6 mL) was loaded into 15 mL tubes to study the effects of HPCD treatment. 10 mL portions of the mixed salmon slurry were loaded into 15 mL tubes for enzymatic hydrolysis under HPCD conditions.

4.1.2 High-pressure carbon dioxide treatment

Prepared samples were divided into two groups. HPCD treatment at 1 MPa was applied to one group according to Japan’s High-Pressure Gas Safety Act, as described in our earlier work (Yu and Iwahashi, 2019), while the other group was not treated using HPCD. A high-pressure vessel (30- 11HF4, High-Pressure Equipment, Erie, PA, USA, volume: 794 cm³) was used for HPCD treatment, and temperature was controlled using a thermostatic bath; Sketch 1 presents a schematic illustration of the setup. A 16 h treatment period was applied to both groups as this accommodated the work-rest schedule of industry workers.

After treatment, the DE was determined using a standard culture medium (0.25 % yeast extract, 0.5 % polypeptone, 0.1 % D (+) -glucose, 1.5 % agar powder, sterilized by autoclaving), as described in a previous study (Yu and Iwahashi, 2019). Spread plates were cultured at 30 °C in an incubator for 3 days followed by colonies counting. The DE was calculated as follows:

42 Sketch 1. The sketch map of high-pressure equipment used in this work. A: CO2 container; B: water bath; C: high pressure vessel (30-11HF4, High Pressure Equipment, Elie, PA, USA, volume 794 cm³) (inside water for transferring heat); D: sample; E: heater; F: temperature keeper.

43 Decontamination efficiency (DE) = (number of colonies in treated sample) / (number of colonies in the pre-treated sample)

4.1.3 DNA extraction and 16S rDNA gene amplification

Following treatment, bacterial DNA was extracted using an Extrap Soil DNA Kit Plus ver. 2 (Nippon Steel Eco-Tech Corporation) using the procedure described in the instruction manual. The extracted DNA was stored at -20 °C for subsequent experiments.

The primers listed in Table 4 (Klindworth et al. 2013) were used to amplify the 16S DNA; this has been widely used for bacterial taxonomy. Tag sequences were used to separate samples. The protocol and PCR conditions used to amplify 16S rDNA were as follows: the 25 μL PCR system contained 12.5 μL of KAPA HiFi HotStart ReadyMix (Nippon Genetics Co., Ltd., Tokyo, Japan), 1.5 μL of a forward primer (5 μM), 1.5 μL of a reverse primer (5 μM), a 2 μL sample, and 7.5 μL of

DNase/RNase-free water. The PCR was run for 35 cycles, including denaturation at 98 °C for 20 s, annealing at 60 °C for 10 s, and extension at 72 °C for 20 s. Initialization was conducted at 95 °C for 3 min, and the final extension was conducted at 72 °C for 2 min. PCR was conducted in a LifeECO cycler (Hangzhou Bioer Technology Co. Ltd.). The amplified DNA libraries were then transferred for subsequent NGS.

4.1.4 Bacterial DNA quantification by real-time PCR and bacterial community analysis by NGS

The total bacterial DNA in samples was quantified using real-time PCR. The extracted DNA was diluted 10 times, and 16S rDNA was amplified as the target gene to compare the bacterial DNA

44 Table 4: The primers information used in this work

Samples Forward primers Reverse primers Tag-F Size Reference

40 ℃ GCGTGACCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC GCGTGA 464bp Klindworth et al., 2013

45 ℃ TAGGACCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC TAGGAC 464bp Klindworth et al., 2013

50 ℃ TTACGTCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC TTACGT 464bp Klindworth et al., 2013

40 ℃_H AGGTCTCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC AGGTCT 464bp Klindworth et al., 2013

45 ℃_H CGTATCCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC CGTATC 464bp Klindworth et al., 2013

50 ℃_H TCAAGCCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC TCAAGC 464bp Klindworth et al., 2013

BT ACCTAGCCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC ACCTAG 464bp Klindworth et al., 2013

Bacteria 16S 5’-AAACTCAAAKGAATTGACGG 5’-CTCACRRCACGAGCTGAC / 136bp Bacchetti et al., 2011

Salmon EF1AB 5’-TGCCCCTCCAGGATGTCTAC 5’-CACGGCCCACAGGTACTG / 59bp Olsvik et al., 2005