Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami
<p>Time-course of protein hydrolysis in the different salami formulations. Protein content was assessed by OPA (<b>A</b>), Coomassie assay (<b>B</b>), and absorbance at 280 nm (<b>C</b>), and it is expressed as milligrams of protein digested/gram of product. Data are means ± SD of in vitro digestion of three independent samples analyzed in triplicate. Protein content is expressed as milligram of protein digested/gram of product. Statistical analysis was by one-way ANOVA (always <span class="html-italic">p</span> < 0.05) with Tukey’s post-hoc test comparing each salami formulation at the three digestion time points (different letters indicate statistical significance). G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase; C-NO<sub>2</sub>: salami with sodium nitrite, potassium nitrate and with nitrate-reducing microbial starter cultures; C-0: salami containing neither nitrate-reducing microbial starter cultures nor additives (nitrite, polyphenols and ascorbate); SA: salami with nitrate-reducing microbial starter cultures and sodium ascorbate; SMA: salami with nitrate-reducing microbial starter cultures, sodium ascorbate and plant extracts.</p> "> Figure 2
<p>Integral area of aliphatic, α-proton, and aromatic amino acid regions at different digestion times in different salamis. Data are means ± SD of in vitro digestion of three independent samples analyzed in duplicate. Integrals of aliphatic (<b>A</b>), α-proton (<b>B</b>), and aromatic (<b>C</b>) amino acid regions are expressed as signal areas. Statistical analysis was by one-way ANOVA (always <span class="html-italic">p</span> < 0.05) with Tukey’s post-hoc test comparing the three digestion times in each salami formulation (different letters indicate significant differences). G120: end of gastric phase; D60: 60 min of duodenal phase; D120: end of duodenal phase; AA: amino acids; C-NO<sub>2</sub>: salami with sodium nitrite, potassium nitrate and with nitrate-reducing microbial starter cultures; C-0: salami containing neither nitrate-reducing microbial starter cultures nor additives (nitrite, polyphenols and ascorbate); SA: salami with nitrate-reducing microbial starter cultures and sodium ascorbate; SMA: salami with nitrate-reducing microbial starter cultures, sodium ascorbate and plant extracts.</p> "> Figure 3
<p>Antiproliferative effect of digested salami formulations. Data are means ± SD of three independent supplementation of in vitro digested, each one analyzed in triplicate. The antiproliferative effect is expressed as the percentage of cell numbers representing unsupplemented (US) cells (assigned 100%). Statistical analysis was by one-way ANOVA (always <span class="html-italic">p</span> < 0.05) with Tukey’s post-hoc test comparing US and supplemented cells for each dilution (different letters indicate significant differences). B: “blank” digestion; dil: dilution; C-NO<sub>2</sub>: salami with sodium nitrite, potassium nitrate and with nitrate-reducing microbial starter cultures; C-0: salami containing neither nitrate-reducing microbial starter cultures nor additives (nitrite, polyphenols and ascorbate); SA: salami with nitrate-reducing microbial starter cultures and sodium ascorbate; SMA: salami with nitrate-reducing microbial starter cultures, sodium ascorbate and plant extracts.</p> "> Figure 4
<p>Genotoxic effect of different salami formulations. Data are means ± SD of three independent supplementation of in vitro digested, each one analyzed in triplicate. The genotoxicity is expressed as tail intensity. Statistical analysis was by one-way ANOVA (<span class="html-italic">p</span> < 0.05) with Tukey’s post-hoc test comparing unsupplemented (US), 2 mM ethylmethanesulfonate (EMS) and digested supplemented cells (different letters indicate significant differences). B: “blank” digestion; C-NO<sub>2</sub>: salami with sodium nitrite, potassium nitrate and with nitrate-reducing microbial starter cultures; C-0: salami containing neither nitrate-reducing microbial starter cultures nor additives (nitrite, polyphenols and ascorbate); SA: salami with nitrate-reducing microbial starter cultures and sodium ascorbate; SMA: salami with nitrate-reducing microbial starter cultures, sodium ascorbate and plant extracts.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Fatty Acid Composition and Bioaccessibility
2.2. Protein Hydrolysis
2.3. HR 1H-NMR Spectroscopy
2.4. Peptides Formation
2.5. Bioinformatic Analysis
2.6. Cell Proliferation and Genotoxicity
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Salami Formulation, Preparation, and Fermentation
4.3. In Vitro Digestion
4.4. Fatty Acids Bioaccessibility
4.5. Protein Hydrolysis
4.6. HR 1H NMR Spectroscopy
4.7. Bioactive Peptides Determination and Identification
4.8. HT-29 Cell Culture and Supplementation
4.9. Cell Proliferation
4.10. Genotoxicity Assay
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Laskowski, W.; Górska-Warsewicz, H.; Kulykovets, O. Meat, Meat Products and Seafood as Sources of Energy and Nutrients in the Average Polish Diet. Nutrients 2018, 10, 1412. [Google Scholar] [CrossRef] [Green Version]
- Rohrmann, S.; Linseisen, J. Processed meat: The real villain? Proc. Nutr. Soc. 2016, 75, 233–241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Crowe, W.; Elliott, C.T.; Green, B.D. A Review of the In Vivo Evidence Investigating the Role of Nitrite Exposure from Processed Meat Consumption in the Development of Colorectal Cancer. Nutrients 2019, 11, 2673. [Google Scholar] [CrossRef] [Green Version]
- Karwowska, M.; Kononiuk, A. Nitrates/Nitrites in Food—Risk for Nitrosative Stress and Benefits. Antioxidants 2020, 9, 241. [Google Scholar] [CrossRef] [Green Version]
- Jo, K.; Lee, S.; Yong, H.I.; Choi, Y.-S.; Jung, S. Nitrite sources for cured meat products. LWT 2020, 129, 109583. [Google Scholar] [CrossRef]
- Balaraman, G.; Sundaram, J.; Mari, A.; Krishnan, P.; Salam, S.; Subramaniam, N.; Sirajduddin, I.; Thiruvengadam, D. Farnesol alleviates diethyl nitrosamine induced inflammation and protects experimental rat hepatocellular carcinoma. Environ. Toxicol. 2021, 36, 2467–2474. [Google Scholar] [CrossRef]
- Kalaycıoğlu, Z.; Erim, F.B. Nitrate and Nitrites in Foods: Worldwide Regional Distribution in View of Their Risks and Benefits. J. Agric. Food Chem. 2019, 67, 7205–7222. [Google Scholar] [CrossRef]
- Zhao, C.; Zhang, H.; Zhou, J.; Lu, Q.; Zhang, Y.; Yu, X.; Wang, S.; Liu, R.; Pu, Y.; Yin, L. Metabolomics-based molecular signatures reveal the toxic effect of co-exposure to nitrosamines in drinking water. Environ. Res. 2022, 204, 111997. [Google Scholar] [CrossRef]
- Asioli, D.; Aschemann-Witzel, J.; Caputo, V.; Vecchio, R.; Annunziata, A.; Næs, T.; Varela, P. Making sense of the “clean label” trends: A review of consumer food choice behavior and discussion of industry implications. Food Res. Int. 2017, 99, 58–71. [Google Scholar] [CrossRef]
- Bedale, W.; Sindelar, J.J.; Milkowski, A.L. Dietary nitrate and nitrite: Benefits, risks, and evolving perceptions. Meat Sci. 2016, 120, 85–92. [Google Scholar] [CrossRef]
- Fraqueza, M.J.; Laranjo, M.; Elias, M.; Patarata, L. Microbiological hazards associated with salt and nitrite reduction in cured meat products: Control strategies based on antimicrobial effect of natural ingredients and protective microbiota. Curr. Opin. Food Sci. 2021, 38, 32–39. [Google Scholar] [CrossRef]
- Munekata, P.E.S.; Pateiro, M.; Domínguez, R.; Pollonio, M.A.R.; Sepúlveda, N.; Andres, S.C.; Reyes, J.; Santos, E.M.; Lorenzo, J.M. Beta vulgaris as a Natural Nitrate Source for Meat Products: A Review. Foods 2021, 10, 2094. [Google Scholar] [CrossRef]
- Jaiswal, A.; Jyothi Lakshmi, A. Maximising the bioaccessibility of iron and zinc of a complementary food mix through multiple strategies. Food Chem. 2022, 372, 131286. [Google Scholar] [CrossRef] [PubMed]
- Mashitoa, F.M.; Akinola, S.A.; Manhevi, V.E.; Garcia, C.; Remize, F.; Slabbert, R.M.; Sivakumar, D. Influence of Fermentation of Pasteurised Papaya Puree with Different Lactic Acid Bacterial Strains on Quality and Bioaccessibility of Phenolic Compounds during In Vitro Digestion. Foods 2021, 10, 962. [Google Scholar] [CrossRef]
- Santos, D.I.; Saraiva, J.M.A.; Vicente, A.A.; Moldão-Martins, M. 2—Methods for determining bioavailability and bioaccessibility of bioactive compounds and nutrients. In Innovative Thermal and Non-Thermal Processing, Bioaccessibility and Bioavailability of Nutrients and Bioactive Compounds; Barba, F.J., Saraiva, J.M.A., Cravotto, G., Lorenzo, J.M., Eds.; Woodhead Publishing: Cambridge, UK, 2019; pp. 23–54. [Google Scholar]
- Shahidi, F.; Pan, Y. Influence of food matrix and food processing on the chemical interaction and bioaccessibility of dietary phytochemicals: A review. Crit. Rev. Food Sci. Nutr. 2022, 62, 6421–6445. [Google Scholar] [CrossRef]
- Di Nunzio, M.; Loffi, C.; Chiarello, E.; Dellafiora, L.; Picone, G.; Antonelli, G.; Di Gregorio, C.; Capozzi, F.; Tedeschi, T.; Galaverna, G.; et al. Impact of a Shorter Brine Soaking Time on Nutrient Bioaccessibility and Peptide Formation in 30-Months-Ripened Parmigiano Reggiano Cheese. Molecules 2022, 27, 664. [Google Scholar] [CrossRef]
- Ponce de León-Rodríguez, M.d.C.; Guyot, J.-P.; Laurent-Babot, C. Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation. Crit. Rev. Food Sci. Nutr. 2019, 59, 3648–3666. [Google Scholar] [CrossRef]
- Herranz, B.; Ordóñez, J.A.; De La Hoz, L.; Hierro, E.; Soto, E.; Cambero, M.I. Fatty acid composition of salami from different countries and their nutritional implications. Int. J. Food Sci. Nutr. 2008, 59, 607–618. [Google Scholar] [CrossRef]
- Egger, L.; Schlegel, P.; Baumann, C.; Stoffers, H.; Guggisberg, D.; Brügger, C.; Dürr, D.; Stoll, P.; Vergères, G.; Portmann, R. Physiological comparability of the harmonized INFOGEST in vitro digestion method to in vivo pig digestion. Food Res. Int. 2017, 102, 567–574. [Google Scholar] [CrossRef]
- Urbinati, E.; Di Nunzio, M.; Picone, G.; Chiarello, E.; Bordoni, A.; Capozzi, F. The Effect of Balsamic Vinegar Dressing on Protein and Carbohydrate Digestibility is Dependent on the Food Matrix. Foods 2021, 10, 411. [Google Scholar] [CrossRef]
- Kotopoulou, S.; Zampelas, A.; Magriplis, E. Dietary nitrate and nitrite and human health: A narrative review by intake source. Nutr. Rev. 2022, 80, 762–773. [Google Scholar] [CrossRef] [PubMed]
- Stoica, M.; Antohi, V.M.; Alexe, P.; Ivan, A.S.; Stanciu, S.; Stoica, D.; Zlati, M.L.; Stuparu-Cretu, M. New Strategies for the Total/Partial Replacement of Conventional Sodium Nitrite in Meat Products: A Review. Food Bioprocess Technol. 2022, 15, 514–538. [Google Scholar] [CrossRef]
- Kramer, B.; Thielmann, J.; Hickisch, A.; Muranyi, P.; Wunderlich, J.; Hauser, C. Antimicrobial activity of hop extracts against foodborne pathogens for meat applications. J. Appl. Microbiol. 2015, 118, 648–657. [Google Scholar] [CrossRef] [PubMed]
- Burri, S.C.M.; Ekholm, A.; Bleive, U.; Püssa, T.; Jensen, M.; Hellström, J.; Mäkinen, S.; Korpinen, R.; Mattila, P.H.; Radenkovs, V.; et al. Lipid oxidation inhibition capacity of plant extracts and powders in a processed meat model system. Meat Sci. 2020, 162, 108033. [Google Scholar] [CrossRef] [PubMed]
- Sallan, S.; Kaban, G.; Şişik Oğraş, Ş.; Çelik, M.; Kaya, M. Nitrosamine formation in a semi-dry fermented sausage: Effects of nitrite, ascorbate and starter culture and role of cooking. Meat Sci. 2020, 159, 107917. [Google Scholar] [CrossRef]
- Gøtterup, J.; Olsen, K.; Knøchel, S.; Tjener, K.; Stahnke, L.H.; Møller, J.K.S. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities. Meat Sci. 2008, 78, 492–501. [Google Scholar] [CrossRef]
- Sánchez Mainar, M.; Leroy, F. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities. Int. J. Food Microbiol. 2015, 212, 60–66. [Google Scholar] [CrossRef]
- Sánchez Mainar, M.; Stavropoulou, D.A.; Leroy, F. Exploring the metabolic heterogeneity of coagulase-negative staphylococci to improve the quality and safety of fermented meats: A review. Int. J. Food Microbiol. 2017, 247, 24–37. [Google Scholar] [CrossRef]
- Lavelli, V.; D’Incecco, P.; Pellegrino, L. Vitamin D Incorporation in Foods: Formulation Strategies, Stability, and Bioaccessibility as Affected by the Food Matrix. Foods 2021, 10, 1989. [Google Scholar] [CrossRef]
- Rousseau, S.; Kyomugasho, C.; Celus, M.; Hendrickx, M.E.G.; Grauwet, T. Barriers impairing mineral bioaccessibility and bioavailability in plant-based foods and the perspectives for food processing. Crit. Rev. Food Sci. Nutr. 2020, 60, 826–843. [Google Scholar] [CrossRef]
- Navarro, J.L.; Nadal, M.I.; Nieto, P.; Flores, J. Effect of nitrate and nitrite curing salts on lipolysis in dry sausages produced using a rapid fermentation process. Z. Für Lebensm. Forsch. A 1998, 206, 217–221. [Google Scholar] [CrossRef]
- Pateiro, M.; Bermúdez, R.; Lorenzo, J.M.; Franco, D. Effect of Addition of Natural Antioxidants on the Shelf-Life of “Chorizo”, a Spanish Dry-Cured Sausage. Antioxidants 2015, 4, 42–67. [Google Scholar] [CrossRef] [Green Version]
- Rysman, T.; Van Hecke, T.; Van Poucke, C.; De Smet, S.; Van Royen, G. Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties. Food Chem. 2016, 209, 177–184. [Google Scholar] [CrossRef] [PubMed]
- Cirkovic Velickovic, T.D.; Stanic-Vucinic, D.J. The Role of Dietary Phenolic Compounds in Protein Digestion and Processing Technologies to Improve Their Antinutritive Properties. Compr. Rev. Food Sci. Food Saf. 2018, 17, 82–103. [Google Scholar] [CrossRef] [Green Version]
- Donadio, G.; Santoro, V.; Dal Piaz, F.; De Tommasi, N. Food Matrices Affect the Peptides Produced during the Digestion of Arthrospira platensis-Based Functional Aliments. Nutrients 2021, 13, 3919. [Google Scholar] [CrossRef]
- Di Nunzio, M.; Picone, G.; Pasini, F.; Caboni, M.F.; Gianotti, A.; Bordoni, A.; Capozzi, F. Olive oil industry by-products. Effects of a polyphenol-rich extract on the metabolome and response to inflammation in cultured intestinal cell. Food Res. Int. 2018, 113, 392–400. [Google Scholar] [CrossRef] [Green Version]
- Paolella, S.; Falavigna, C.; Faccini, A.; Virgili, R.; Sforza, S.; Dall’Asta, C.; Dossena, A.; Galaverna, G. Effect of dry-cured ham maturation time on simulated gastrointestinal digestion: Characterization of the released peptide fraction. Food Res. Int. 2015, 67, 136–144. [Google Scholar] [CrossRef]
- Bauchart, C.; Morzel, M.; Chambon, C.; Mirand, P.P.; Reynès, C.; Buffière, C.; Rémond, D. Peptides reproducibly released by in vivo digestion of beef meat and trout flesh in pigs. Br. J. Nutr. 2007, 98, 1187–1195. [Google Scholar] [CrossRef] [Green Version]
- Dziuba, J.; Minkiewicz, P.; Nałecz, D.; Iwaniak, A. Database of biologically active peptide sequences. Food Nahr. 1999, 43, 190–195. [Google Scholar] [CrossRef]
- Escudero, E.; Sentandreu, M.A.; Arihara, K.; Toldrá, F. Angiotensin I-Converting Enzyme Inhibitory Peptides Generated from in Vitro Gastrointestinal Digestion of Pork Meat. J. Agric. Food Chem. 2010, 58, 2895–2901. [Google Scholar] [CrossRef]
- Li, G.-H.; Le, G.-W.; Shi, Y.-H.; Shrestha, S. Angiotensin I–converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutr. Res. 2004, 24, 469–486. [Google Scholar] [CrossRef]
- Chai, K.F.; Voo, A.Y.H.; Chen, W.N. Bioactive peptides from food fermentation: A comprehensive review of their sources, bioactivities, applications, and future development. Compr. Rev. Food Sci. Food Saf. 2020, 19, 3825–3885. [Google Scholar] [CrossRef] [PubMed]
- Gallego, M.; Mora, L.; Escudero, E.; Toldrá, F. Bioactive peptides and free amino acids profiles in different types of European dry-fermented sausages. Int. J. Food Microbiol. 2018, 276, 71–78. [Google Scholar] [CrossRef] [PubMed]
- Simpson, D.J. Proteolytic degradation of cereal prolamins—The problem with proline. Plant Sci. 2001, 161, 825–838. [Google Scholar] [CrossRef]
- Ansari, F.A.; Ali, S.N.; Arif, H.; Khan, A.A.; Mahmood, R. Acute oral dose of sodium nitrite induces redox imbalance, DNA damage, metabolic and histological changes in rat intestine. PLoS ONE 2017, 12, e0175196. [Google Scholar] [CrossRef] [Green Version]
- Grudziński, I. Studies on the mechanism of the toxic action of sodium nitrite on intestinal absorption in rats. Arch. Environ. Contam. Toxicol. 1991, 21, 475–479. [Google Scholar] [CrossRef]
- Yu, J.; Wang, Y.; Xiao, Y.; Li, X.; Xu, X.; Zhao, H.; Wu, L.; Li, J. Effects of chronic nitrate exposure on the intestinal morphology, immune status, barrier function, and microbiota of juvenile turbot (Scophthalmus maximus). Ecotoxicol. Environ. Saf. 2021, 207, 111287. [Google Scholar] [CrossRef]
- Di Nunzio, M.; Picone, G.; Pasini, F.; Chiarello, E.; Caboni, M.F.; Capozzi, F.; Gianotti, A.; Bordoni, A. Olive oil by-product as functional ingredient in bakery products. Influence of processing and evaluation of biological effects. Food Res. Int. 2020, 131, 108940. [Google Scholar] [CrossRef] [Green Version]
- Hosseini, F.; Majdi, M.; Naghshi, S.; Sheikhhossein, F.; Djafarian, K.; Shab-Bidar, S. Nitrate-nitrite exposure through drinking water and diet and risk of colorectal cancer: A systematic review and meta-analysis of observational studies. Clin. Nutr. 2021, 40, 3073–3081. [Google Scholar] [CrossRef]
- Tiso, M.; Schechter, A.N. Nitrate reduction to nitrite, nitric oxide and ammonia by gut bacteria under physiological conditions. PLoS ONE 2015, 10, e0119712. [Google Scholar] [CrossRef]
- Wang, S.; Li, X.; Wang, W.; Zhang, H.; Xu, S. Application of transcriptome analysis: Oxidative stress, inflammation and microtubule activity disorder caused by ammonia exposure may be the primary factors of intestinal microvilli deficiency in chicken. Sci. Total Environ. 2019, 696, 134035. [Google Scholar] [CrossRef] [PubMed]
- Saccani, G.; Bergamaschi, M.; Schivazappa, C.; Cirlini, M.; Galaverna, G.; Virgili, R. Evaluation of the antioxidant effect of a phytocomplex addition in clean label pork salami enriched in n-3 PUFA. Food Chem. 2023, 399, 133963. [Google Scholar] [CrossRef]
- Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carrière, F.; Boutrou, R.; Corredig, M.; Dupont, D.; et al. A standardised static in vitro digestion method suitable for food—An international consensus. Food Funct. 2014, 5, 1113–1124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Folch, J.; Lees, M.; Stanley, G.H.S. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957, 226, 497–509. [Google Scholar] [CrossRef]
- Stoffel, W.; Chu, F.; Ahrens, E.H. Analysis of Long-Chain Fatty Acids by Gas-Liquid Chromatography. Anal. Chem. 1959, 31, 307–308. [Google Scholar] [CrossRef]
- Dima, C.; Assadpour, E.; Dima, S.; Jafari, S.M. Bioavailability and bioaccessibility of food bioactive compounds; overview and assessment by in vitro methods. Compr. Rev. Food Sci. Food Saf. 2020, 19, 2862–2884. [Google Scholar] [CrossRef]
- Bradstreet, R.B. Kjeldahl Method for Organic Nitrogen. Anal. Chem. 1954, 26, 185–187. [Google Scholar] [CrossRef]
- Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Church, F.C.; Porter, D.H.; Catignani, G.L.; Swaisgood, H.E. An o-phthalaldehyde spectrophotometric assay for proteinases. Anal Biochem. 1985, 146, 343–348. [Google Scholar] [CrossRef]
- Minkiewicz, P.; Iwaniak, A.; Darewicz, M. BIOPEP-UWM Database of Bioactive Peptides: Current Opportunities. Int. J. Mol. Sci. 2019, 20, 5978. [Google Scholar] [CrossRef]
- Kumar, R.; Chaudhary, K.; Sharma, M.; Nagpal, G.; Chauhan, J.S.; Singh, S.; Gautam, A.; Raghava, G.P.S. AHTPDB: A comprehensive platform for analysis and presentation of antihypertensive peptides. Nucleic Acids Res. 2014, 43, D956–D962. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cordelli, E.; Bignami, M.; Pacchierotti, F. Comet assay: A versatile but complex tool in genotoxicity testing. Toxicol. Res. 2021, 10, 68–78. [Google Scholar] [CrossRef] [PubMed]
CNO2 | C0 | SA | SMA | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
G120 | D60 | D120 | G120 | D60 | D120 | G120 | D60 | D120 | G120 | D60 | D120 | |
14:0 | 0.5 ± 0.7 c | 30.6 ± 4.2 b | 37.3 ± 0.2 a | 0.3 ± 0.2 b | 38.0 ± 6.5 a | 43.6 ± 6.4 a | 0.4 ± 0.1 b | 37.7 ± 1.8 a | 42.1 ± 3.1 a | 0.7 ± 0.4 b | 38.6 ± 2.4 a | 40.0 ± 0.6 a |
16:0 | 0.8 ± 0.4 c | 29.2 ± 3.0 b | 37.4 ± 2.7 a | 0.4 ± 0.2 b | 39.2 ± 8.3 a | 45.6 ± 7.9 a | 1.0 ± 0.1 c | 32.3 ± 1.1 b | 43.9 ± 4.2 a | 0.9 ± 0.5 b | 40.2 ± 2.2 a | 37.5 ± 0.0 a |
16:1 n−7 | 0.4 ± 0.5 b | 27.0 ± 4.7 a | 31.7 ± 0.3 a | 0.0 ± 0.0 b | 32.6 ± 5.7 a | 37.1 ± 5.7 a | 0.0 ± 0.0 b | 29.8 ± 3.1 a | 37.9 ± 4.8 a | 0.1 ± 0.1 b | 35.0 ± 2.8 a | 38.2 ± 2.5 a |
18:0 | 0.9 ± 0.6 b | 28.2 ± 1.2 a | 38.4 ± 8.5 a | 0.6 ± 0.1 b | 40.2 ± 8.9 a | 47.3 ± 0.8 a | 1.2 ± 0.1 c | 30.8 ± 1.0 b | 43.5 ± 4.6 a | 1.0 ± 0.3 c | 30.4 ± 2.2 b | 40.3 ± 1.8 a |
18:1 n−9 | 0.6 ± 0.4 b | 31.0 ± 4.7 a | 37.5 ± 0.6 a | 0.2 ± 0.1 b | 37.2 ± 7.9 a | 43.1 ± 6.6 a | 0.8 ± 0.0 c | 33.0 ± 2.7 b | 42.3 ± 4.0 a | 0.6 ± 0.5 c | 39.0 ± 2.4 b | 41.8 ± 2.8 a |
18:2 n−6 | 0.6 ± 0.5 c | 36.6 ± 3.8 b | 45.2 ± 3.0 a | 0.3 ± 0.1 b | 39.9 ± 8.7 a | 47.2 ± 6.3 a | 0.4 ± 0.1 c | 42.4 ± 5.4 b | 54.4 ± 6.2 a | 0.6 ± 0.4 b | 44.0 ± 3.5 a | 48.1 ± 2.1 a |
18:3 n−3 | 0.0 ± 0 b | 40.3 ± 14.0 a | 45.2 ± 4.5 a | 0.6 ± 1.1 b | 38.7 ± 19.7 a | 51.2 ± 6.1 a | 0.0 ± 0.0 b | 41.2 ± 8.1 a | 51.2 ± 4.1 a | 0.0 ± 0.0 b | 44.3 ± 6.7 a | 49.8 ± 1.1 a |
20:1 n−9 | 0.0 ± 0 b | 22.1 ± 7.5 a | 29.3 ± 0.8 a | 0.0 ± 0 b | 30.1 ± 7.3 a | 38.1 ± 5.1 a | 0.0 ± 0.0 b | 25.0 ± 2.5 a | 35.6 ± 5.8 a | 0.0 ± 0.0 b | 30.4 ± 0.8 a | 33.4 ± 3.6 a |
20:4 n−6 | 0.0 ± 0 b | 46.3 ± 3.2 a | 70.3 ± 17.3 a | 0.0 ± 0 b | 53.0 ± 16.7 a | 70.0 ± 14.4 a | 0.0 ± 0.0 b | 49.5 ± 12.3 a | 70.3 ± 14.6 a | 0.0 ± 0.0 c | 45.0 ± 2.3 b | 50.8 ± 3.2 a |
Total | 0.6 ± 0.4 c | 30.7 ± 3.8 b | 38.3 ± 2.5 a | 0.33 ± 0.1 b | 38.3 ± 8.2 a | 44.7 ± 7.4 a | 0.8 ± 0.0 c | 33.3 ± 1.3 b | 44.1 ± 3.6 a | 0.7 ± 0.4 b | 39.9 ± 2.4 a | 39.8 ± 1.3 a |
Protein Source (UNIPROT) | Type | CNO2 | ANOVA | C0 | ANOVA | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
ND | G120 | D60 | D120 | p Value | ND | G120 | D60 | D120 | p Value | ||
Actin (P68137) | MF | 12.0 ± 0.0 a | 25.5 ± 6.4 a | 10.0 ± 4.2 a | 11.5 ± 0.7 a | <0.05 | 12.0 ± 0 b | 22.5 ± 0.7 a | 9.5 ± 2.1 b | 14.0 ± 1.4 b | <0.05 |
Myosin I (Q9TV61) | MF | 4.0 ± 0.0 a | 0.5 ± 0.7 a | 1.5 ± 2.1 a | 2.0 ± 1.4 a | n.s. | 4.0 ± 0 a | 2.5 ± 0.7 ab | 0.5 ± 0.7 b | 2.5 ± 0.7 b | <0.05 |
Myosin II (Q9TV63) | MF | 1.0 ± 0.0 a | 0.0 ± 0.0 a | 0.5 ± 0.7 a | 0.5 ± 0.7 a | n.s. | 1.0 ± 0 a | 0.0 ± 0.0 a | 0.5 ± 0.7 a | 1.0 ± 0.0 a | n.s. |
Myosin IV (Q9TV62) | MF | 0.0 ± 0.0 a | 3.0 ± 1.4 a | 7.0 ± 4.2 a | 6.5 ± 2.1 a | n.s. | 0.0 ± 0.0 b | 3.5 ± 0.7 b | 4.5 ± 2.1 b | 11.0 ± 1.4 a | <0.05 |
Myosin VI (Q29122) | MF | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 3.0 ± 0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Myosin VII (P79293) | MF | 14.0 ± 0.0 a | 1.0 ± 0 a | 11.0 ± 4.2 a | 13.5 ± 6.4 a | n.s. | 14.0 ± 0 a | 3.0 ± 1.4 c | 8.5 ± 2.1 b | 19.0 ± 0 a | <0.05 |
Myosin I LC (A1XQT6) | MF | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 3.0 ± 0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Myosin VII HC (K7GMH0) | MF | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Tropomyosin α-1 chain (F2Z5B6) | MF | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 5.0 ± 0 a | 3.5 ± 0.7 b | <0.05 | 0.0 ± 0.0 a | 0.0 ± 0.0 a | 3.0 ± 1.4 a | 4.5 ± 0.7 a | <0.05 |
Troponin T (Q75NG6) | MF | 1.0 ± 0 a | 0 ± 0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
PK (F1SHL9) | SP | 3.0 ± 0 a | 1.0 ± 0 c | 3.0 ± 0 a | 2.0 ± 0 b | <0.05 | 3.0 ± 0.0 a | 1.0 ± 0 a | 4.0 ± 2.8 a | 3.5 ± 0.7 a | n.s. |
Albumin (P008835) | SP | 1.0 ± 0.0 a | 0.5 ± 0.7 a | 0.5 ± 0.7 a | 1.0 ± 0 a | n.s. | 1.0 ± 0 a | 0 ± 0 a | 0.5 ± 0.7 a | 0.5 ± 0.7 a | n.s. |
FBA (F1RJ25) | SP | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0 ± 0 b | <0.05 | 1.0 ± 0 a | 0 ± 0 b | 0 ± 0 b | 0.0 ± 0.0 b | <0.05 |
PBK (F1RP07) | SP | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | < 0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
NI | 5.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | < 0.05 | 5.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | |
MF peptides | 39.0 ± 0.0 a | 30.0 ± 5.7 a | 35.0 ± 15.6 a | 37.5 ± 6.4 a | n.s. | 39.0 ± 0.0 a | 31.5 ± 3.5 a | 26.5 ± 9.2 b | 52.0 ± 4.2 a | n.s. | |
SP peptides | 6.0 ± 0.0 a | 1.5 ± 0.7 b | 3.5 ± 0.7 b | 3.0 ± 0.0 b | < 0.05 | 6.0 ± 0.0 a | 1.0 ± 0.0 b | 4.5 ± 2.1 ab | 4.0 ± 0.0 ab | <0.05 | |
Total peptides | 50.0 ± 0.0 a | 31.5 ± 4.9 a | 38.5 ± 16.3 a | 40.5 ± 6.4 a | n.s. | 50.0 ± 0.0 ab | 32.5 ± 3.5 bc | 31.0 ± 7.1 c | 56.0 ± 4.2 a | <0.05 | |
Average peptides lengths (in AA) | 11.0 ± 0.0 a | 12.0 ± 0.0 a | 6.0 ± 0.0 b | 6.0 ± 0.0 b | <0.05 | 11.0 ± 0.0 a | 12.5 ± 0.7 a | 6.0 ± 0.0 b | 6.0 ± 0.0 b | <0.05 | |
Protein source (UNIPROT) | Type | SA | ANOVA | SMA | ANOVA | ||||||
ND | G120 | D60 | D120 | p value | ND | G120 | D60 | D120 | p value | ||
Actin (P68137) | MF | 12.0 ± 0.0 b | 23.5 ± 3.5 a | 10.0 ± 1.4 b | 11.0 ± 1.4 b | <0.05 | 12.0 ± 0.0 b | 23.0 ± 1.4 a | 12.5 ± 2.1 b | 14.0 ± 0.0 b | <0.05 |
Myosin I (Q9TV61) | MF | 4.0 ± 0.0 a | 0.5 ± 0.7 a | 1.0 ± 1.4 a | 1.0 ± 1.4 a | n.s. | 4.0 ± 0.0 a | 2.0 ± 0.0 a | 1.0 ± 1.4 a | 1.5 ± 2.1 a | n.s. |
Myosin II (Q9TV63) | MF | 1.0 ± 0.0 a | 0.5 ± 0.7 a | 0.5 ± 0.7 a | 1.0 ± 0.0 a | n.s. | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 1.0 ± 0.0 a | 1.0 ± 0.0 a | <0.05 |
Myosin IV (Q9TV62) | MF | 0.0 ± 0.0 c | 2.5 ± 0.7 bc | 7.0 ± 0.0 a | 5.0 ± 1.4 ab | <0.05 | 0.0 ± 0.0 a | 2.5 ± 0.7 a | 6.0 ± 1.4 a | 8.5 ± 4.9 a | n.s. |
Myosin VI (Q29122) | MF | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Myosin VII (P79293) | MF | 14.0 ± 0.0 ab | 0.5 ± 0.7 b | 11.5 ± 6.4 ab | 15.5 ± 3.5 ab | <0.05 | 14.0 ± 0.0 a | 1.5 ± 0.7 b | 12.0 ± 0.0 a | 15.0 ± 4.2 a | <0.05 |
Myosin I LC (A1XQT6) | MF | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 3.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Myosin VII HC (K7GMH0) | MF | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
Tropomyosin α-1 chain (F2Z5B6) | MF | 0.0 ± 0.0 a | 0.0 ± 0.0 a | 3.0 ± 2.8 a | 4.5 ± 2.1 a | n.s. | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 4.0 ± 1.4 a | 3.5 ± 0.7 a | <0.05 |
Troponin T (Q75NG6) | MF | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
PK (F1SHL9) | SP | 3.0 ± 0.0 a | 1.5 ± 0.7 a | 1.0 ± 0.0 a | 4.5 ± 2.1 a | n.s. | 3.0 ± 0.0 a | 3.0 ± 1.4 a | 4.0 ± 1.4 a | 4.0 ± 1.4 a | n.s. |
Albumin (P008835) | SP | 1.0 ± 0.0 a | 0.5 ± 0.7 a | 1.0 ± 0.0 a | 0.5 ± 0.7 a | n.s. | 1.0 ± 0.0 a | 1.0 ± 0.0 a | 0.5 ± 0.7 a | 0.5 ± 0.7 a | n.s. |
FBA (F1RJ25) | SP | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
PBK (F1RP07) | SP | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 1.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 |
NI | 5.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 5.0 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | |
MF peptides | 39.0 ± 0.0 a | 27.5 ± 2.1 a | 33.0 ± 12.7 a | 38.0 ± 9.9 a | n.s. | 39.0 ± 0.0 a | 29.0 ± 0.0 a | 36.5 ± 2.1 a | 43.5 ± 12.0 a | n.s. | |
SP peptides | 6.0 ± 0.0 a | 2.0 ± 0.0 a | 2.0 ± 0.0 a | 5.5 ± 2.1 a | <0.05 | 6.0 ± 0.0 a | 4.0 ± 1.4 a | 4.5 ± 0.7 a | 4.5 ± 0.0 a | n.s. | |
Total peptides | 50.0 ± 0.0 a | 29.5 ± 2.1 a | 35.0 ± 12.7 a | 43.5 ± 7.8 a | n.s. | 50.0 ± 0.0 a | 33.0 ± 1.4 a | 41.0 ± 1.4 a | 48.0 ± 11.3 a | n.s. | |
Average peptides lengths (in AA) | 11.0 ± 0.0 a | 11.0 ± 0.0 a | 6.0 ± 0.0 b | 6.0 ± 0.0 b | <0.05 | 11.0 ± 0.0 a | 11.5 ± 0.7 a | 6.0 ± 0.0 b | 6.0 ± 0.0 b | <0.05 |
Protein Sequence | Protein Source | CNO2 | ANOVA p Value | C0 | ANOVA p Value | Reported Activity (µM IC50) | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
ND | G120 | D60 | D120 | ND | G120 | D60 | D120 | |||||
FQPSF | Actin (P68137) | 0.0 ± 0.0 b | 2.9 ± 0.5 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 0.0 ± 0.0 b | 2.7 ± 0.2 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | ACE inhibitor (12.6) |
AGDDAPRAVF | Actin (P68137) | 0.0 ± 0.0 b | 2.6 ± 0.3 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 0.0 ± 0.0 b | 2.9 ± 0.1 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | Bitterness suppressing (n.r.) |
AGDDAPR | Actin (P68137) | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 2.7 ± 0.0 b | 3.1 ± 0.2 a | <0.05 | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 3.8 ± 0.0 a | 2.9 ± 0.3 b | <0.05 | Antioxidant (n.r.), ACE (11.9), pancreatic lipase (110.6), and α-amylase (14.7) inhibitor |
VAPEEHPT | Actin (P68137) | 0.0 ± 0.0 b | 0.0 ± 0.0 b | 3.0 ± 0.3 a | 3.1 ± 0.3 a | <0.05 | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 3.7 ± 0.5 a | 2.7 ± 0.0 b | <0.05 | DPP-IV inhibitor (n.r.) |
Protein sequence | Protein source | SA | ANOVA p value | SMA | ANOVA p value | Reported activity (µM IC50) | ||||||
ND | G120 | D60 | D120 | ND | G120 | D60 | D120 | |||||
FQPSF | Actin (P68137) | 0.0 ± 0.0 b | 3.4 ± 0.1 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | 0.0 ± 0.0 b | 3.1 ± 0.1 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | ACE inhibitor (12.6) |
AGDDAPRAVF | Actin (P68137) | 0.0 ± 0.0 a | 0.0 ± 0.0 a | 0.0 ± 0.0 a | 0.0 ± 0.0 a | n.s. | 0.0 ± 0.0 b | 2.9 ± 0.0 a | 0.0 ± 0.0 b | 0.0 ± 0.0 b | <0.05 | Bitterness suppressing (n.r.) |
AGDDAPR | Actin (P68137) | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 4.3 ± 0.3 a | 3.4 ± 0.1 b | <0.05 | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 3.7 ± 0.0 a | 2.8 ± 0.1 b | <0.05 | Antioxidant (n.r.), ACE (11.9), pancreatic lipase (110.6), and α-amylase (14.7) inhibitor |
VAPEEHPT | Actin (P68137) | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 3.6 ± 0.2 a | 3.2 ± 0.1 b | <0.05 | 0.0 ± 0.0 c | 0.0 ± 0.0 c | 3.1 ± 0.0 a | 2.7 ± 0.0 b | <0.05 | DPP-IV inhibitor (n.r.) |
Ingredient (g) | CNO2 | C0 | SA | SMA |
---|---|---|---|---|
Lean muscle tissue | 750 | 750 | 750 | 750 |
Fat muscle tissue | 250 | 250 | 250 | 250 |
Salt | 25 | 25 | 25 | 25 |
Sugar | 2 | 2 | 2 | 0 |
Spice mix 1 | 0.3 | 0.3 | 0.3 | 0.3 |
Sodium ascorbate | 0.5 | 0 | 0.5 | 0.5 |
Sodium nitrite | 0.05 | 0 | 0 | 0 |
Potassium nitrate | 0.08 | 0 | 0 | 0 |
Polyphenol mix 2 | 0 | 0 | 0 | 0.86 |
Coagulase-negative Staphylococcaceae | 0.25 | 0 | 0.25 | 0.25 |
Lactic acid Bacteria | 0.125 | 0 | 0.125 | 0.125 |
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Di Nunzio, M.; Loffi, C.; Montalbano, S.; Chiarello, E.; Dellafiora, L.; Picone, G.; Antonelli, G.; Tedeschi, T.; Buschini, A.; Capozzi, F.; et al. Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami. Int. J. Mol. Sci. 2022, 23, 12555. https://doi.org/10.3390/ijms232012555
Di Nunzio M, Loffi C, Montalbano S, Chiarello E, Dellafiora L, Picone G, Antonelli G, Tedeschi T, Buschini A, Capozzi F, et al. Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami. International Journal of Molecular Sciences. 2022; 23(20):12555. https://doi.org/10.3390/ijms232012555
Chicago/Turabian StyleDi Nunzio, Mattia, Cecilia Loffi, Serena Montalbano, Elena Chiarello, Luca Dellafiora, Gianfranco Picone, Giorgia Antonelli, Tullia Tedeschi, Annamaria Buschini, Francesco Capozzi, and et al. 2022. "Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami" International Journal of Molecular Sciences 23, no. 20: 12555. https://doi.org/10.3390/ijms232012555