Meat peptone and meat extract were purchased from Merck, Darmstadt, Germany. 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT), Tetracycline (≥98.0% (NT)) and Sephadex LH-20 were purchased from Sigma-Aldrich, St. Louis, MO, USA. All materials were used as received.
Meat peptone
Manufactured by Merck Group
Sourced in Germany, Ireland
Meat peptone is a nutrient-rich powder derived from the enzymatic hydrolysis of meat protein. It serves as a growth medium component for the cultivation of various microorganisms in laboratory settings.
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Lab products found in correlation
Meat extract, by Merck Group (3 mentions)
Yeast extract, by Merck Group (3 mentions)
Peptone from soybean, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Potato dextrose agar (pda), by Biolife (2 mentions)
2 4 iodophenyl 3 4 nitrophenyl 5 phenyl 2h tetrazolium chloride, by Merck Group (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Sodium dihydrogen phosphate monohydrate, by Merck Group (1 mentions)
Starch, by Merck Group (1 mentions)
L glutamic acid γ monohydroxamate, by Merck Group (1 mentions)
P nitrophenyl butyrate, by Merck Group (1 mentions)
Maltose, by Merck Group (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Coomassie blue g 250, by Merck Group (1 mentions)
4 aminoantipyrine, by Merck Group (1 mentions)
Calcium chloride, by Kemika (1 mentions)
3 5 dinitrosalicylic acid, by Merck Group (1 mentions)
Folin ciocalteu s phenol reagent, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
10 protocols using meat peptone
1
Quantitative Tetrazolium Reduction Assay
2
Comprehensive Chemical Reagent Inventory
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Chemicals including 100% acetic acid, 4-aminoantipyrine, Coomassie Blue G-250, ethanol, ferric chloride, hydrogen chloride, meat extract, meat peptone, n-butanol, 85% phosphoric acid, potassium dihydrogen phosphate, sodium chloride, sodium dihydrogen phosphate monohydrate, and sodium hydrogen phosphate were from Merck, Darmstadt, Germany. Acetonitrile, agar, amylose-remazol brilliant blue R, bovine serum albumin, casein, 3,5-dinitrosalicylic acid, 2,2-diphenyl-1-picrylhydrazyl, Folin-Ciocalteu’s phenol-reagent, gallic acid, hydrochloric acid, hydrogen peroxide, 99% hydroxylamine hydrochloride, L-glutamic acid γ-monohydroxamate, L- Glutathione reduced, maltose, methanol, yeast extract, peptone from soybean, phenol, p-nitrophenyl butyrate, potassium sodium tartrate tetrahydrate, sodium acetate, sodium carbonate, starch, trichloroacetic acid, and Trizma Base were obtained from Sigma-Aldrich, Taufkirchen, Germany. CBZ-Glutaminylglycine was purchased from Zedira GmbH, Darmstadt, Germany. Potato dextrose agar and Mueller-Hinton broth were purchased from Biolife, Milano, Italy. Calcium chloride, D-(+)-glucose anhydrous, and iron (II) sulfate heptahydrate were obtained at Kemika, Zagreb, Croatia, while malt extract, potato dextrose broth, Triton X-100, tryptic soy broth, and tryptone were purchased from Fluka, Buchs, Switzerland.
3
Raman Spectroscopy of Bacterial Cultures
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For this study, the food spoilage-relevant bacteria were cultivated on a nutrient agar with the composition of 18 g/L agar-agar, 10 g/L meat extract, 10 g/L meat peptone and 5 g/L sodium chloride (Merck KGaA, Darmstadt, Germany). The bacteria were as follows:
Escherichia coli K12 DSM 498, TOP10, and HB101; Micrococcus luteus DSM 20030; Brochothrix thermosphacta DSM 20171 (B. therm); Pseudomonas fluorescens (Ps. fluor) DSM 4358 and DSM 50090; Bacillus subtilis DSM 10 (B. sub); and Bacillus thuringiensis israelensis DSM 5724 (B. tii).
The microbial samples to be examined were taken directly from the medium by means of a swab through a stainless steel cylinder without further sample preparation [40 (link),55 (link)]. Raman spectra of samples that were cultivated under lifetime stress conditions were recorded immediately after sampling. Otherwise, the samples were subjected to sampling stress and examined spectroscopically without further incubation time after the stress impact.
Escherichia coli K12 DSM 498, TOP10, and HB101; Micrococcus luteus DSM 20030; Brochothrix thermosphacta DSM 20171 (B. therm); Pseudomonas fluorescens (Ps. fluor) DSM 4358 and DSM 50090; Bacillus subtilis DSM 10 (B. sub); and Bacillus thuringiensis israelensis DSM 5724 (B. tii).
The microbial samples to be examined were taken directly from the medium by means of a swab through a stainless steel cylinder without further sample preparation [40 (link),55 (link)]. Raman spectra of samples that were cultivated under lifetime stress conditions were recorded immediately after sampling. Otherwise, the samples were subjected to sampling stress and examined spectroscopically without further incubation time after the stress impact.
4
Cultivation and Spectral Profiling of Microbial Strains
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The following nine microorganisms were cultivated on nutrient agar (10 g/L meat peptone, 10 g/L meat extract, 5 g/L sodium chloride and 18 g/L agar-agar (Merck KGaA, Germany)) and prepared according to our previously published method [12 (link),37 ,38 (link)]: Bacillus subtilis DSM 10, Bacillus coagulans DSM 1, Escherichia coli K12 DSM 498, Escherichia coli TOP10, Micrococcus luteus DSM 20030, Brochothrix thermosphacta DSM 20171, Pseudomonas fluorescens DSM 4358, Pseudomonas fluorescens DSM 50090 and Bacillus thuringiensis israelensis DSM 5724.
As described in our previously published methods [12 (link)], the samples were taken by a blotting technique with the sample carrier (stainless steel cylinder) directly from the agar plate without any sampling pre-treatments (e.g., centrifugation, washing, drying). IR spectra were recorded directly (lifetime conditions) after sampling or directly after the stress impact (sampling condition) without any incubation period after sampling. Detailed information can be found inSection 2.2 .
The spectral data set of each microorganism, consisting of four independent data sets for each stress condition, were divided into independent training and test data sets. Further information can be found inSection 2.4 .
As described in our previously published methods [12 (link)], the samples were taken by a blotting technique with the sample carrier (stainless steel cylinder) directly from the agar plate without any sampling pre-treatments (e.g., centrifugation, washing, drying). IR spectra were recorded directly (lifetime conditions) after sampling or directly after the stress impact (sampling condition) without any incubation period after sampling. Detailed information can be found in
The spectral data set of each microorganism, consisting of four independent data sets for each stress condition, were divided into independent training and test data sets. Further information can be found in
5
Cultivation Media Ingredients Sourcing
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Peptone from soybean, yeast extract, and agar were obtained from Sigma-Aldrich, St. Luis, MO, USA. Sodium chloride, ethanol, meat extract, and meat peptone were obtained from Merck, Darmstadt, Germany. Triptic soy broth, tryptone, potato dextrose broth, and malt extract were obtained from Fluka, Buchs, Switzerland. Potato dextrose agar was from Biolife, Milano, Italy, while anhydrous D-(+)-glucose was obtained from Kemika, Zagreb, Croatia.
6
Synthetic Urban Wastewater Preparation
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The chemicals used for the preparation of synthetic urban wastewater were glucose (C6H12O6) D(+) glucose anhydrous, extra pure, Ph Eur, BP, USP (Sigma-Aldrich; Baden-Württemberg, Germany); meat peptone (Sigma-Aldrich; Baden-Württemberg, Germany); urea (Urea, ACS reagent grade, Sigma-Aldrich; Baden-Württemberg, Germany); sodium chloride (NaCl reagent grade, ACS, ISO, Reag. Ph Eur, Sigma-Aldrich; Baden-Württemberg, Germany); sodium bicarbonate (NaHCO3, extra pure, Pharmpure®, Ph Eur, BP, USP, Sigma-Aldrich; Baden-Württemberg, Germany); di-potassium hydrogen phosphate anhydrous (K2HPO4 for analysis, ExpertQ®, ACS, Reag. Ph Eur, Sigma-Aldrich; Baden-Württemberg, Germany); calcium chloride dihydrate (CaCl2·2H2O powder, for analysis, ExpertQ®, ACS, Sigma-Aldrich; Baden-Württemberg, Germany); magnesium sulfate heptahydrate (MgSO4·7H2O for analysis, ExpertQ®, ACS, Reag. Ph Eur Sigma-Aldrich, Baden-Württemberg, Germany); and iron (III) chloride hexahydrate, (FeCl3·6H2O ACS reagent 97%, Sigma Aldrich, Baden-Württemberg, Germany). The chemicals used for the membrane recycling process and membrane cleaning were sodium hypochlorite (NaOCl 10% w/v, Scharlab; Barcelona, Spain) and ethanol (96% EPR Ph.Eur. Sigma-Aldrich; Baden-Württemberg, Germany). Samples and solutions were prepared using Milli-Q water.
7
Thermoresistant Protease Production from Pseudomonas
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Pseudomonas fluorescens ATCC 17556 was grown in nutrient broth (meat extract 3 g/L, meat peptone 5 g/L; Sigma Aldrich, Dublin, Ireland) for 72 h at 26°C with stirring at 90 rpm. Then, 1 mL of the inoculated nutrient broth was spread plated on blood agar plates (horse blood agar, 7% concentration, Base No. 2; Oxoid, Basingstoke, UK) and plates were incubated for 24 h at 26°C. Bacterial colonies were transferred to 100 mL of sterilized reconstituted skim milk powder (10% wt/vol). The inoculated reconstituted skim milk bottles were incubated for 120 h at 10°C with stirring at 90 rpm. Then, the bottles were centrifuged at 20,000 × g at 25°C for 30 min, and the supernatant containing protease was collected. The same supernatant containing the protease was used in a previous study conducted by the authors, and it was determined that the protease was thermoresistant, because it remained active in the supernatant after being heated at 72°C for 15 s (temperature applied during pasteurization; Paludetti et al., 2020) (link).
8
Protease Production from Pseudomonas fluorescens
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Pseudomonas fluorescens (ATCC 17556) was grown in nutrient broth (meat extract 3 g/L, meat peptone 5 g/L; Sigma Aldrich, Dublin, Ireland) for 48 h at 26°C with stirring at 90 rpm. Afterward, 1 mL of the inoculated nutrient broth was spread-plated on blood agar plates (horse blood agar, 7% concentration, base No. 2, Oxoid, Basingstoke, UK) and plates were incubated for 24 h at 26°C. Bacterial colonies were transferred to 100 mL of sterilized reconstituted skim milk powder (RSMP; 10% wt/vol). The inoculated RSMP bottles were incubated for 120 h at 10°C with stirring at 90 rpm. Afterward, RSMP was centrifuged at 20,000 × g at ambient temperature for 30 min and the supernatant containing protease was collected.
9
Enzymatic Hydrolysis of Chicken By-products and Spruce Wood
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Fresh chicken by-products (heart, liver and digestive tract) were provided by Nortura Hærland (Hærland, Norway) and kept at − 20 °C until further use. Prior to the enzymatic hydrolysis reactions, samples were thawed and minced with a BIRO® MODEL 6642 feed grinder (Marblehead, Ohio, USA). The chicken by-products contained 30.12 ± 0.50% dry matter, including 15.10 ± 1.20% protein, 4.47 ± 0.29% ash and 6.91 ± 0.55% lipids [20 (link)]. Enzymatic hydrolysates of BALI™ pretreated spruce wood (Picea abies) were kindly provided by Borregaard AS (Sarpsborg, Norway) [16 (link)]. The raw material used in the pulping process was chipped spruce with chip sizes of up to 4.5 × 4.5 × 0.8 cm3. The carbohydrate composition of the spruce hydrolysate is shown in Additional file 1 : Table S1. Yeast extract, meat peptone, yeast nitrogen base w/o amino acids and w/o ammonium sulfate, glucose, cellobiose, xylose, lactic acid, acetic acid, sulfuric acid, hydrogen chloride, sodium hydroxide, ninhydrin, glycine and stannous chloride were purchased from Sigma-Aldrich (Missouri, USA). ammonium sulfate was purchased from VWR (Pennsylvania, USA), and urea was kindly provided by Yara International ASA (Oslo, Norway). Kjeltabs for Kjeldahl analysis were purchased from Thomson and Capper Ltd. (Cheshire, UK).
10
Glycerol and Peptone Gavage in Rat Metabolism
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A C C E P T E D M A N U S C R I P T 6 22mg glycerol/ml, Fresenius Kabi, UK) or meat peptone (0.5g/ml dissolved in water, Sigma Aldrich, UK). Thirty minutes after gavage, AIN-93M stock diet was supplied for 2h. Food not consumed was presented at ZT11 for 2h. The quantity of food consumed was measured by weighing the food that remained at the end of the 2h access period. Twenty four hours later, the gavage was repeated but food was withheld at the scheduled feed. Ninety minutes after gavage, the rats were euthanized by anaesthetising with isoflurane, followed by decapitation. Trunk blood was collected into 15ml polypropylene tubes containing 50 I.U. heparin and 8mM DPPIV inhibitor (KR-62436 hydrate, Sigma Aldrich, UK) and stored on ice until processed for plasma. Brains were removed and frozen on dry ice and stored at -80°C until use. Blood was centrifuged at 1000g for 15 minutes at 4°C and the plasma removed to microfuge tubes and stored in aliquots at -80°C.
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