Effect of salt concentrations on <i>in vitro</i> rumen fermentation of cellulose, starch, and protein

The aim of this study was to evaluate the effects of various concentrations of three salts (sodium chloride (NaCl), magnesium chloride (MgCl2), and calcium chloride (CaCl2)) on the in vitro rumen fermentation of cellulose, starch, and protein substrates. Six salt concentrations were tested, separately, namely 0, 100, 200, 400, 800, and 1600 mg/dL. The experiment was conducted using the completely randomized design in a 6 × 3 × 3 factorial arrangement with main effects of salt concentration and salt type (six levels of three salts (NaCl, MgCl2, or CaCl2) (0, 100, 200, 400, 800, and 1600 mg/dL) into three substrates [starch, cellulose, and glucose]) with three replicates. Cellulose- and glucose-fermenting bacteria were sensitive to NaCl concentrations greater than 400 mg/dL (17.48 decisiemens per metre (dS/m)) and 800 mg/dL (20.55 dS/m) in the media, respectively. In contrast, starch-fermenting bacteria continued to grow in NaCl concentrations up to 1600 mg/dL (29.09 dS/m). Thus, it was concluded that starch-fermenting microorganisms tolerated higher concentrations of NaCl compared with the other microbial groups. Cellulose-fermenting microorganisms are less tolerant to MgCl2 in relation to the other microbial groups. Starch, cellulose-, and glucose-fermenting bacteria from cattle tolerate CaCl2 concentrations of up to 1600 mg/dL (12.26 dS/m). These results suggest that brackish water may be used for ruminants. However, it is important perform an analysis of that water and then to adjust diets to minimize the effects of types of salt and concentrations of salt on rumen microorganisms. Keywords: brackish water, dissolved salts, rumen microbes, water quality

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Effect of spineless-cactus mucilage on the in vitro rumen fermentation of cellulose, starch, and protein

Revista Brasileira de Saúde e Produção Animal ◽

10.1590/s1519-99402017000400002 ◽

2017 ◽

Vol 18 (4) ◽

pp. 505-517 ◽

Cited By ~ 1

Author(s):

Ricardo Martins Araujo Pinho ◽

Edson Mauro Santos ◽

Juliana Silva de Oliveira ◽

Alexandre Henrique Remigio Loureiro ◽

Alberto Jefferson da Silva Macêdo ◽

...

Keyword(s):

Protein A ◽

Rumen Fermentation ◽

Rumen Microorganisms ◽

Randomized Experimental Design ◽

The Media ◽

Butyrate Fermentation ◽

Cactus Mucilage ◽

The Individual ◽

In Vitro Rumen Fermentation

SUMMARY The aim of this study was to evaluate the effect of the levels of spineless-cactus mucilage on the in vitro rumen fermentation of cellulose, starch, and protein. A completely randomized experimental design was adopted with a 5 × 3 factorial arrangement consisting of five levels of spineless-cactus mucilage (0, 5, 10, 20, and 40%) and three substrates (carboxymethylcellulose, starch, and trypticase). Treatments were evaluated in a ruminal environment simulated by in vitro incubation at different times of assessment: 0, 3, 6, 12, 24, and 48 h. The incubation procedure was repeated three times, totaling three evaluations per incubation time for each treatment. There was an interaction (P<0.05) between the mucilage levels and substrate for all evaluated ruminal parameters, except for the concentration of microbial protein after 48 h of fermentation and for the proportions of acetate and butyrate fermentation at time 0 h. There was a quadratic increase (P<0.05) in the concentration of ammoniacal nitrogen after 48 h of incubation in the media containing carboxymethylcellulose and trypticase. pH values decreased quadratically (P<0.05) as a function of the mucilage levels in the media containing carboxymethylcellulose and trypticase. Overall, no expressive alterations were observed between the individual molar proportions of acetate, propionate, and butyrate with the addition of spineless-cactus mucilage levels to the different substrates. Spineless- cactus mucilage affects the pattern of fermentation of starch, cellulase, and protein performed by rumen microorganisms.

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The Antimethanogenic Nitrocompounds Can be Cleaved into Nitrite by Rumen Microorganisms: A Comparison of Nitroethane, 2-Nitroethanol, and 2-Nitro-1-propanol

Metabolites ◽

10.3390/metabo10010015 ◽

2019 ◽

Vol 10 (1) ◽

pp. 15

Author(s):

Zhen-Wei Zhang ◽

Yan-Lu Wang ◽

Wei-Kang Wang ◽

Yong-Yang Chen ◽

Xue-Meng Si ◽

...

Keyword(s):

Compartment Model ◽

Rumen Fermentation ◽

Rumen Microorganisms ◽

Batch Cultures ◽

Rumen Microbes ◽

Fermentation Pattern ◽

Maize Meal ◽

The One

A class of aliphatic short chain nitrocompounds have been reported as being capable of CH4 reduction both in vitro and in vivo. However, the laboratory evidence associated with the metabolic fate of nitrocompounds in the rumen has not been well documented. The present study was conducted to compare in vitro degradation and metabolism of nitroethane (NE), 2-nitroethanol (NEOH), and 2-nitro-1-propanol (NPOH) incubated with mixed rumen microorganisms of dairy cows. After 10 mM supplementation of nitrocompounds, a serious of batch cultures were carried out for 120 h under the presence of two substrates differing in the ratio of maize meal to alfalfa hay (HF, 1:4; LF, 4:1). Compared to the control, methane production was reduced by 59% in NPOH and by >97% in both NE and NEOH, and such antimethanogenic effects were more pronounced in the LF than the HF group. Although NE, NEOH, and NPOH addition did not alter total VFA production, the rumen fermentation pattern shifted toward increasing propionate and butyrate and decreasing acetate production. The kinetic disappearance of each nitrocompound was well fitted to the one-compartment model, and the disappearance rate (k, %/h) of NE was 2.6 to 5.2 times greater than those of NEOH and NPOH. Higher intermediates of nitrite occurred in NEOH in comparison with NPOH and NE while ammonia N production was lowest in NEOH. Consequently, a stepwise accumulation of bacterial crude protein (BCP) in response to the nitrocompound addition was observed in both the HF and LF group. In brief, both NE and NEOH in comparison with NPOH presented greater antimethanogenic activity via the shift of rumen fermentation. In addition, the present study provided the first direct evidence that rumen microbes were able to cleave these nitrocompounds into nitrite, and the subsequent metabolism of nitrite into ammonia N may enhance the growth of rumen microbes or promote microbial activities.

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Isolation and Characterization of Yeasts from Rumen Fluids for Potential Use as Additives in Ruminant Feeding

Veterinary Sciences ◽

10.3390/vetsci8030052 ◽

2021 ◽

Vol 8 (3) ◽

pp. 52

Author(s):

Chanon Suntara ◽

Anusorn Cherdthong ◽

Metha Wanapat ◽

Suthipong Uriyapongson ◽

Vichai Leelavatcharamas ◽

...

Keyword(s):

Incubation Time ◽

Cellulase Activity ◽

The Other ◽

Pichia Kudriavzevii ◽

Factor B ◽

Yeast Strains ◽

Randomized Design ◽

Holstein Friesian ◽

The Media ◽

Completely Randomized Design

Saccharomyces cerevisiae is a yeast strain often used to improve the feed quality of ruminants. However, S. cerevisiae has limited capacity to provide biomass when inoculated with carbon sources and a low ability to produce cellulase enzymes. Here, we hypothesized that yeast in the rumen produces a large amount of biomass and could release cellulase enzymes to break down fiber content. Therefore, the aim of this study was to screen, isolate and identify yeast from the rumen fluids of Holstein Friesian steers and measure the efficiency of biomass production and cellulase activity. A fermentation medium containing sugarcane molasses as a carbon source and urea as a nitrogen source was optimized. Two fistulated–crossbred Holstein Friesian steers averaging 350 ± 20 kg body weight were used to screen and isolate the ruminal yeast. Two experiments were designed: First, a 12 × 3 × 3 factorial was used in a completely randomized design to determine biomass and carboxymethyl cellulase activity. Factor A was the isolated yeast and S. cerevisiae. Factor B was sugarcane molasses (M) concentration. Factor C was urea (U) concentration. In the second experiment, potential yeasts were selected, identified, and analyzed for 7 × 4 factorial use in a completely randomized design. Factor A was the incubation times. Factor B was the isolated yeast strains, including codes H-Khon Kaen University (KKU) 20 (as P. kudriavzevii-KKU20), I-KKU20 (C. tropicalis-KKU20), and C-KKU20 (as Galactomyces sp.-KKU20). Isolation was imposed under aerobic conditions, resulting in a total of 11 different colonies. Two appearances of colonies including asymmetric colonies of isolated yeast (indicated as A, B, C, E, and J) and ovoid colonies (coded as D, F, G, H, I, and K) were noted. Isolated yeast from the rumen capable of providing a high amount of biomass when inoculant consisted of the molasses 15% + urea 3% (M15 + U3), molasses 25% + urea 1% (M25 + U1), molasses 25% + urea 3% (M25 + U3), and molasses 25% + urea 5% (M25 + U5) when compared to the other media solution (p < 0.01). In addition, 11 isolated biomass-producing yeasts were found in the media solution of M25 + U1. There were 4 isolates cellulase producing yeasts discovered in the media solution of M25 + U1 and M25 + U5 whereas molasses 5% + urea 1% (M5 + U1), molasses 5% + urea 3% (M5 + U3), molasses 5% + urea 5% (M5 + U5), molasses 15% + urea 1% (M15 + U1), molasses 15% + urea 3% (M5 + U3), and M25 + U3 were found with 2, 3, 1, 2, 1, and 2 isolates, respectively. Ruminal yeast strains H-KKU20, I-KKU20, and C-KKU20 were selected for their ability to produce biomass. Identification of isolates H-KKU20 and I-KKU20 revealed that those isolates belonged to Pichia kudriavzevii-KKU20 and Candida tropicalis-KKU20 while C-KKU20 was identified as Galactomyces sp.-KKU20. Two strains provided maximum cell growth: P. kudriavzevii-KKU20 (9.78 and 10.02 Log cell/mL) and C. tropicalis-KKU20 (9.53 and 9.6 Log cells/mL) at 60 and 72 h of incubation time, respectively. The highest ethanol production was observed in S. cerevisiae at 76.4, 77.8, 78.5, and 78.6 g/L at 36, 48, 60, and 72 h of incubation time, respectively (p < 0.01). The P. kudriavzevii-KKU20 yielded the least reducing sugar at about 30.6 and 29.8 g/L at 60 and 72 h of incubation time, respectively. The screening and isolation of yeasts from rumen fluids resulted in 11 different yeasts being obtained. The potential yeasts discovered in the rumen fluid of cattle were Pichia kudriavzevii-KKU20, Candida tropicalis-KKU20, and Galactomyces sp.-KKU20. P. kudriavzevii-KKU20 had higher results than the other yeasts in terms of biomass production, cellulase enzyme activity, and cell number.

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Rumen fermentation kinetics as impacted by red amaranth (Amaranthus cruentus, L) leaf powder using in vitro gas production technique

10.21203/rs.3.rs-20200/v1 ◽

2020 ◽

Author(s):

Thiwakorn Ampapon ◽

Bounnaxay Viennasay ◽

Metha Wanapat

Keyword(s):

Rumen Fermentation ◽

Gas Production ◽

Amaranthus Cruentus ◽

Randomized Design ◽

In Vitro Gas Production ◽

Two Factors ◽

Dry Matter Degradation ◽

Leaf Powder ◽

Completely Randomized Design

Abstract Background A need for research searching for alternative rumen enhancers warrants immediate attention. The in vitro fermentation experiment was conducted using factorial arrangement of two factors of roughage to concentrate and seven level of red amaranth leaf powder percentage of total substrate in a Completely randomized design (CRD). Two factors, namely Factor A was two ratio of roughage (R) to concentrate (C) at 60:40 and 40:60 and Factor B was level of red amaranth (Amaranthus cruentus, L) leaf powder (RALP) supplementation at 0, 2, 4, 6, 8, 10, and 12% of total dietary substrate. Results Red amaranth leaf powder (RALP) contained phytonutrients both condensed tannins and saponins in addition with high macro minerals (Ca, K, and Mg). This experiment revealed innovations of the RALP supplementation by enhancing rumen propionate (C3) production, reducing acetate (C2) to (C3) ratio, reducing protozoal population and mitigating methane (CH4) production. Furthermore, rumen dry matter degradation percentages were remarkably enhanced (P < 0.001) by increasing RALP supplementation. Conclusion Plants rich in phytonutrients and minerals such as red amaranth leaf powder (RALP) have a vital and promising role in modulating rumen fermentation, mitigating methane production, as well as increasing substrate degradability.

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Study of the effects of PR toxin, mycophenolic acid and roquefortine C on in vitro gas production parameters and their stability in the rumen environment

The Journal of Agricultural Science ◽

10.1017/s0021859614000343 ◽

2014 ◽

Vol 153 (1) ◽

pp. 163-176 ◽

Cited By ~ 12

Author(s):

A. GALLO ◽

G. GIUBERTI ◽

T. BERTUZZI ◽

M. MOSCHINI ◽

F. MASOERO

Keyword(s):

Mycophenolic Acid ◽

Rumen Fluid ◽

Rumen Fermentation ◽

Gas Production ◽

Farm Animals ◽

Marginal Effect ◽

Rumen Microorganisms ◽

Marginal Effects ◽

Roquefortine C

SUMMARYMoulds belonging to Penicillium section roqueforti are common contaminants of feedstuffs and produce several mycotoxins that can cause health hazards when ingested by farm animals. Among these, PR toxin (PR), mycophenolic acid (MY) and roquefortine C (RC) have been frequently detected in forages, particularly silages. The aims of the current trials were to study the effects of the presence of pure mycotoxins on in vitro rumen fermentation parameters and to assess their stability in the rumen environment. Two successive in vitro gas production experiments were carried out: a central composite design with four replications of central point (CCD) and a completely randomized design with a fully factorial arrangement of treatments (FFD). In CCD, the effects of PR, MY and RC concentrations in diluted rumen fluid (i.e. 0·01, 0·30, 1·01, 1·71 and 2·00 μg of each mycotoxin/ml) were tested. Gas volume produced after 48 h of incubation (Vf) decreased linearly as concentrations of RC and MY in diluted rumen fluid increased, with marginal effects similar for two mycotoxins, being respectively −14·6 and −13·4 ml/g organic matter (OM) for each 1·0 μg/ml of increment in mycotoxin concentration. Similarly, total volatile fatty acid (VFA) production decreased quadratically as concentrations of RC and MY increased, with marginal effects about two times higher for MY than RC, being −4·22 and −2·62 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration. With respect to maximum Vf (i.e. 410·6 ml/g OM) and VFA (98·06 mmol/l) values estimated by the model, decreases of 13·6 and 15·2% were obtained when incubating the highest RC and MY concentrations, respectively. The PR did not interfere with rumen fermentation pattern and it was not recovered after 48 h of incubation, whereas the stabilities of MY and RC in rumen fluid were similar and on average equal to about 50%. On the basis of CCD results, a second experiment (FFD) was carried out in which only effects of MY and RC concentrations (i.e. 0, 0·67, 1·33 and 2·00 μg of each mycotoxin/ml of diluted rumen fluid) were tested. Data from FFD showed Vf decreased linearly when concentrations of MY and RC increased, with marginal effect two-folds higher for MY than for RC (−11·1 ml/g OM and −6·7 ml/g OM, respectively). Similar marginal effects of MY and RC in decreasing VFA production were recorded: −2·38 and −2·86 mmol/l for each 1·0 μg/ml of increment in mycotoxin concentration, respectively. At the highest RC and MY tested concentrations, Vf and VFA decreased by 8·7 and 10·7%, respectively, over maximum estimated values. In FFD, the average amounts of MY and RC recovered in rumen fluid after 48 h of incubation were 79·0 and 40·6%, respectively. In conclusion, the MY and RC from standards interfered with rumen microorganisms at relatively low levels and were partially stable in the rumen environment after 48 h of incubation. These findings suggested that MY and RC could interfere with digestive processes and might represent a potential risk for ruminants fed diets containing feeds contaminated by mycotoxins produced by P. roqueforti.

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The effect of essential oils of Zataria multiflora and Mentha spicata on the in vitro rumen fermentation, and growth and deaminative activity of amino acid-fermenting bacteria isolated from Mehraban sheep

Animal Production Science ◽

10.1071/an12244 ◽

2014 ◽

Vol 54 (3) ◽

pp. 299 ◽

Cited By ~ 2

Author(s):

M. Taghavi-Nezhad ◽

D. Alipour ◽

M. D. Flythe ◽

P. Zamani ◽

G. Khodakaramian

Keyword(s):

Essential Oils ◽

Rumen Fermentation ◽

Gas Production ◽

Feed Additives ◽

Rrna Gene ◽

Ammonia Production ◽

Mentha Spicata ◽

Zataria Multiflora ◽

Rumen Microbes

Gas (CO2 and CH4) and ammonia production in the rumen represent major sources of lost carbon and nitrogen, respectively. The essential oils of some plants have been shown to decrease gas and ammonia production by selectively inhibiting rumen microbes. Particularly, those of Zataria multiflora (ZEO; thymol 21%, carvacrol 32%) and Mentha spicata (SEO; carvone 55%) were evaluated in vitro as ruminant-feed additives. The experiments employed mixed rumen microbes and a hyper-ammonia-producing bacterium (HAP) isolated from the rumen of a Mehraban sheep. Both ZEO and SEO decreased in vitro fibre digestibility and also gas production by mixed rumen microbes that were fermenting a typical growing-lamb diet. ZEO decreased ammonia concentration in mixed culture of rumen microbes, but SEO exerted the opposite effect. A bacterial isolate (MT8) was obtained from the rumen of a Mehraban sheep, and the 16S rRNA gene sequence indicated that it was most closely related to Clostridium bifermentans. Isolate MT8 exhibited rapid ammonia production when peptides were the growth substrate, which indicated that MT8 was a HAP. Both oils inhibited the growth and ammonia production of isolate MT8. However, ZEO decreased ammonia production at lower doses, and to a greater degree, than did SEO. These results indicated that both essential oils could potentially be used to modulate rumen fermentation. The detrimental effects on fibre digestion could be problematic in high-forage diets, and this requires further investigation. Isolate MT8 is the first described HAP from the Mehraban sheep rumen. Results on ammonia production by isolate MT8 and mixed rumen microbes indicate differential mode of action of each oil on this parameter.

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EFFECTS OF THIAMINE INHIBITORS ON CELLULOSE DIGESTION AND VOLATILE FATTY ACID PRODUCTION IN ARTIFICIAL RUMEN FERMENTATIONS

Canadian Journal of Animal Science ◽

10.4141/cjas65-019 ◽

1965 ◽

Vol 45 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

L. P. Milligan ◽

J. M. Asplund ◽

A. R. Robblee

Keyword(s):

Fatty Acid ◽

Acid Production ◽

Volatile Fatty Acid ◽

Fermentation Medium ◽

Rumen Fermentation ◽

Fatty Acid Production ◽

Rumen Microorganisms ◽

Cellulose Digestion ◽

Structural Analogues

The effect of the inclusion of thiamine inhibitors in a rumen fermentation medium on cellulose digestion and volatile fatty acid production by rumen microorganisms in vitro was studied. The addition of the structural analogues oxythiamine and neopyrithiamine to the fermentation medium at concentrations up to 4000 μM and 2000 μM, respectively, did not influence cellulose digestion or volatile fatty acid production. The addition of thiopental and hexetidine to the fermentation medium affected both cellulose digestion and volatile fatty acid production. Some inhibition of cellulose digestion and of volatile fatty acid production occurred with concentrations of thiopental and hexetidine in the fermentation medium as low as 720 μM and 15 μM respectively.

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Experiments on rumen retention time, fermentation rate and dry-matter digestibility in zebu and european-type cattle on a grass hay ration

The Journal of Agricultural Science ◽

10.1017/s0021859600021377 ◽

1960 ◽

Vol 54 (3) ◽

pp. 417-420 ◽

Cited By ~ 15

Author(s):

G. D. Phillips ◽

R. E. Hungate ◽

A. MacGregor ◽

D. P. Hungate

Keyword(s):

Retention Time ◽

Dry Matter ◽

Rumen Fermentation ◽

The Other ◽

Fermentation Products ◽

Fermentation Rate ◽

Dry Matter Digestibility ◽

Two Factors ◽

High Level

1. Experiments are described in which retention time of digesta in the reticulo-rumen, fermentation rates of rumen contents, and dry-matter digestibilities were studied simultaneously in four grade European and three zebu steers.2. Fermentation rates and rumen retentions were significantly negatively correlated.3. Correlations between digestibility and the other two factors were not significant at a high level.4. The multiple regressions calculated for retention time and fermentation rate were significant at the 5% level and that for digestibility approached this level.5. While only fermentation rates show significant differences for the two types of cattle, the results suggest that grades and zebus differ also in the rate of passage of digesta through the rumen.6. The loss in weight of substrate per unit of fermentation products was measured inin vitroexperiments.7. Using certain assumptions, estimates are made of the extent to which the measured fermentation rates could account for the loss in weight of dry matter during digestion, and are compared with the loss actually found.

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Relationship between rumen odd and branched chain fatty acids and fermentation characteristics as studied in vitro

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200013107 ◽

2003 ◽

Vol 2003 ◽

pp. 151-151

Author(s):

B. Vlaeminck ◽

V. Fievez ◽

H. van Laar ◽

D. Demeyer

Keyword(s):

Fatty Acids ◽

Volatile Fatty Acids ◽

Rumen Fermentation ◽

Rumen Microbes ◽

Branched Chain Fatty Acids ◽

Fermentation Pattern ◽

Branched Chain ◽

On Farm ◽

Chain Fatty Acids

Rumen microbes contain a high proportion (20 to 50%) of their fatty acids (FA) as odd and branched chain fatty acids (OBCFA; C15:0, iso C15:0, anteiso C15:0, C17:0; iso C17:0; anteiso C17:0 and C17:1) and different bacterial classes have distinctive OBCFA ‘fingerprints’. As OBCFA make up around 5% of FA in milk, it has been suggested that there is scope for these compounds to be used in on-farm diagnostic milk-based tests in relation to the rumen fermentation pattern. Correlations of milk OBCFA with rumen fermentation pattern were recently shown (Vlaeminck et al., 2002). In the current in vitro study, the potential of rumen OBCFA to predict the production of volatile fatty acids (VFA) was evaluated.

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In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio

Frontiers in Nutrition ◽

10.3389/fnut.2021.772645 ◽

2022 ◽

Vol 8 ◽

Author(s):

Zhi Yuan Ma ◽

Ju Wang Zhou ◽

Si Yu Yi ◽

Min Wang ◽

Zhi Liang Tan

Keyword(s):

Bacterial Diversity ◽

Volatile Fatty Acids ◽

Rumen Fluid ◽

Starch Content ◽

Block Design ◽

Rumen Fermentation ◽

Rumen Microorganisms ◽

Fermentative Bacteria ◽

In Vitro Inoculation

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

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