Heat-stable α-amylase (Termamyl 120 L, 120 KNU-S/mL), protease (Alcalase 2.4 L, 4.42 AU/mL), and amyloglucosidase (AMG 300 L, 300 AGU/mL) were purchased from Novozymes (Beijing, China) biotechnology Co., Ltd. Folin—Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine (TPTZ), 3′,6′-dihydroxy-spiro[isobenzofuran-1(3H),9′-(9H)-xanthene]-3-one disodium salt (FL) and 2,2′-azobis-(2-amidinopropane) dihydrochloride (ABAP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Gallic acid, chlorogenic acid, protocatechuic acid, p-hydroxybenzonic acid, vanillic acid, catechin, epicatechin, caffeic acid, sinapic acid, syringic acid, vanilline, isoquercitrin, p-coumaric acid, ferulic acid, quercetin, caffeic acid methyl, ferulic acid methyl were purchased from Aladdin Reagents (Shanghai, China). Fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Atlanta Biologicals (Lawrenceville, GA, USA), GibcoLife Technologies (Grand Island, NY, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. HepG2 human liver cancer cells were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). All other chemicals used were of analytical grade or above.
Termamyl 120 l
Manufactured by Novozymes
Sourced in Denmark
Termamyl 120 L is a laboratory-scale enzyme product manufactured by Novozymes. It is a thermostable alpha-amylase enzyme that catalyzes the hydrolysis of starch.
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Lab products found in correlation
Amg 300 l, by Novozymes (5 mentions)
Te 149, by Tecnal (2 mentions)
2 2 azobis 2 amidinopropane dihydrochloride abap, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Atlanta Biologicals (1 mentions)
2 4 6 tripyridyl s triazine, by Merck Group (1 mentions)
6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid, by Merck Group (1 mentions)
Amyloglucosidase, by Novozymes (1 mentions)
Hepg2 human liver cancer cells, by American Type Culture Collection (1 mentions)
Pepsin, by Merck Group (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Deuterated nmr solvents, by Merck Group (1 mentions)
Driselase, by Merck Group (1 mentions)
Alcalase 2.5 l, by Novozymes (1 mentions)
Amyloglucosidase amg 300 l, by Novozymes (1 mentions)
Neutrase 0.8 l, by Novozymes (1 mentions)
Alcalase 2, by Novozymes (1 mentions)
Gc 2010, by Shimadzu (1 mentions)
9 protocols using termamyl 120 l
1
Enzymatic Hydrolysis and Antioxidant Assays
2
Dietary Fiber Quantification by Enzymatic Method
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The contents of TDF, consisting of soluble dietary fibre (SDF) and insoluble dietary fibre (IDF) were estimated using enzymatic method [18 (link)]. The assumption in this method is to determine the content of dietary fibre under conditions similar to those found in the human alimentary tract using the following enzymes: thermostable α-amylase (Termamyl 120 L, pH 6.0, 90°C, 15 min.)- Novozymes, Bagsvaerd, Denmark; pepsin (pH 1.5, 40°C, 1 h) and pancreatin (pH 6.8, 40°C, 1 h)- Sigma-Aldrich, Seelze, Germany. Following the enzymatic extraction, the samples were washed with 3 × 20 mL of hot water, 3 × 10 mL of 96 % ethanol and 3 × 20 mL of acetone (Poch, Gliwice, Poland, pure p.a.). Filters with the residue (IDF) were dried at 135°C for 2 h and then incinerated for 5 h in an oven at 525°C. In order to determine SDF the filtrate was mixed with 96 % ethanol (400 mL, 60°C) and left for 2 h. The precipitated dietary fibre was washed with 3 × 20 mL of hot water, 3 × 10 mL of 96 % ethanol and 3 × 20 mL of acetone, and then the filters with the residue (SDF) were dried at 135°C for 2 h and incinerated in an oven at 525°C for 5 h. Analyses were performed using a Fibertec System 1023 apparatus (Foss, Sweden).
3
Enzymatic Hydrolysis of Polysaccharides
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Heat‐stable α‐amylase Termamyl 120 L (from Bacillus licheniformis, 120 KNU/g), the protease Alcalase 2.5 L (from Bacillus licheniformis, 2.5 AU/g), and the amyloglucosidase AMG 300 L (from Aspergillus niger, 300 AGU/g) were kindly donated by Novozymes (Bagsvaerd, Denmark); the complex carbohydrase mixture Driselase (from Basidiomycetes sp.) was from Sigma‐Aldrich (St. Louis, MO). Chemicals used, including deuterated NMR solvents, were either from Sigma‐Aldrich, Roth (Karlsruhe, Germany), VWR (Radnor, PA, USA), or Alfa Aesar (Ward Hill, MA, USA).
4
Isolation and Characterization of Diverse Cereal Fibers
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Cellulose was from Roth, xylans (from beechwood) were purchased from Sigma. Maize (Zea mays L.) middlings were a gift from Cornexo GmbH (Freimersheim, Germany). Intermediate wheatgrass (Thinopyrum intermedium) grain was kindly shared by Dr. Lee DeHaan from The Land Institute, Salina, Kansas, USA. Wholegrain maize flour (Zea mays L.) was a gift from Mühle Beck (Keltern, Germany). Wild rice (Zizania aquatica L.), long-grain brown rice (Oryza sativa L.), rye (Secale cereale L.), kamut (Triticum turanicum Jakubz.), wheat (Triticum aestivum L.), spelt (Triticum spelta L.), popcorn (Zea mays L. var. everta), oat (Avena sativa L.) (dehulled), barley (Hordeum vulgare L.) (dehulled), and proso millet (Panicum miliaceum L.) whole grains were purchased from local grocery stores. Thermostable α-amylase (Termamyl 120 L), amyloglucosidase (AMG 300 L), and protease (Alcalase 1.5 MG Type FG) for preparative fiber isolation were kind gifts from Novozymes (Bagsvaerd, Denmark).
5
Comprehensive Feedstock Analysis Protocol
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Feed samples were dried in a forced ventilation oven at 55 °C for 72 h, ground to 1-mm size in a Wiley mill, and analyzed according to AOAC (1990) as follows: dry matter (DM), method 967.03, ash, method 942.05, crude protein (CP), method 981.10 and EE, method 920.29. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed using detergent solution (Van Soest, 1994 ) using thermostable amylase (Termamyl 120 L Novozymes A/S, Bagsvaerd, Denmark) and without sodium sulfite in a fiber extractor (Tecnal, TE-149; Tecnal, Piracicaba, SP, Brazil). NDF and ADF were not corrected for residual protein and ash. The concentration of total digestible nutrients (TDN) were calculated according to Weiss et al. (1992) .
6
Screening Lactic Acid Bacteria for EPS Production
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Among 5 lactic acid bacteria, the EPS producing-lactic acid bacteria were screened by formation of a viscous substance on the colony and were anaerobically incubated in 1% sucrose-MRS medium at 37°C. After the mass culture, the viscous substance (supernatant, 6 mL) produced by lactic acid bacteria was collected by centrifugation (1,000 rpm for 2 min at 4°C) and mixed vigorously with 2 volumes of 100% EtOH. The precipitate was collected by centrifugation at 4,000 rpm for 10 min at 4°C.
For analysis of sugar composition, a precipitate was hydrated with sterile distilled water (6 mL) and was separated by HPLC without enzymatic treatment and with fructanase- and glucanase-treatment. For treatment of fructanase, the hydrated sample (2 mL) was incubated with invertase (10 μL, Novozymes, Bagsværd, Denmark) at 30°C for 16 h. In case of glucanase, the hydrated sample (2 mL) was incubated with liquefying Termamyl® 120 L (17 μL, Novozymes) at 80°C for 2 h, and then was incubated further with Neutrase 0.8 L (50 μL, Novozymes) at 37°C for 30 min, and β-amylase (17 μL, Amg-300L, Novozymes) at 60°C for 2 h.
For analysis of sugar composition, a precipitate was hydrated with sterile distilled water (6 mL) and was separated by HPLC without enzymatic treatment and with fructanase- and glucanase-treatment. For treatment of fructanase, the hydrated sample (2 mL) was incubated with invertase (10 μL, Novozymes, Bagsværd, Denmark) at 30°C for 16 h. In case of glucanase, the hydrated sample (2 mL) was incubated with liquefying Termamyl® 120 L (17 μL, Novozymes) at 80°C for 2 h, and then was incubated further with Neutrase 0.8 L (50 μL, Novozymes) at 37°C for 30 min, and β-amylase (17 μL, Amg-300L, Novozymes) at 60°C for 2 h.
7
Recombinant TK-PUL Production Protocol
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The chemicals used in the study were of high grade and purchased from Sigma-Aldrich or Fluka, if not mentioned otherwise. Escherichia coli BL21-CodonPlus (DE3)-RIL strain was used as expression host, while recombinant plasmid Pul-pET was used for production of recombinant TK-PUL [3] (link). Industrially employed liquefying amylase, TERMAMYL ® 120L, and saccharifying amyloglucosidase, AMG 300L, were from Novozymes.
8
Enzymatic Hydrolysis of Starch
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Heat stable α-amylase Termamyl 120 L (from Bacillus licheniformis, 120 KNU/g), the protease Alcalase 2.5 (from B. licheniformis, 2.5 AU/g), and the amyloglucosidase AMG 300 L (from Aspergillus niger, 300 AGU/g) were kindly donated by Novozymes (Bagsvaerd, Denmark). Chemicals used were purchased either from VWR, Part of Avantor, Sigma-Aldrich, Alfa Aesar, or Roth.
9
Analytical Methods for Feed and Feces Composition
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The analysis of the chemical composition of feeds, orts, and feces was conducted according to AOAC (12) as follows: DM -method 967,03; CP-method 981,10; Ashesmethod 942,05; and EEmethod 920,29. The NDF content was analyzed in a Tecnal TE-149® fiber analyzer (Tecnal, Piracicaba, SP, Brazil) using 5 X 5 cm non-woven fabric (NWF) bags with 100 μm porosity. To these were added 0.5 g of sample (feed or faeces) and followed for neutral detergent analysis according to the methodology of Van Soest et al (13) without sodium sulfite and using thermostable amylase (Termamyl 120 L Novozymes A/S, Bagsvaerd, Denmark). Subsequently, the NDF was corrected for ashes and the aNDFom content was calculated. The same procedure used for the NDF was used to analyze the material resulting from the in situ ruminal degradation but without the use of amylase and correction for ashes. The NSC content was calculated as proposed by Sniffen et al (10) with the equation: NSC= 100 -(CP + ashes + aNDFom + EE).
The N-NH3 content analysis used the supernatant of ruminal liquid samples thawed at 4 ºC and distillation with 2N KOH according to Ribeiro et al (14) . The concentration of VFAs was determined by gas chromatography (Shimadzu GC-2010, Kyoto, Japan) according to the methodology described by Erwin et al (15) .
The N-NH3 content analysis used the supernatant of ruminal liquid samples thawed at 4 ºC and distillation with 2N KOH according to Ribeiro et al (14) . The concentration of VFAs was determined by gas chromatography (Shimadzu GC-2010, Kyoto, Japan) according to the methodology described by Erwin et al (15) .
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