extfieldsofstudy: Medicine Chemistry

text:
Cellulase recycling in high-solids enzymatic hydrolysis of pretreated empty fruit bunches

Background The lignocellulosic biomass feedstocks such as empty fruit bunches (EFBs) prove to be potential renewable resources owing to their abundance, low prices, and high carbohydrate contents. Generally, the conversion of lignocellulosic biomass into chemicals, fuels, and materials mainly includes pretreatment, enzymatic hydrolysis, fermentation, and recovery of final products. To increase the economic viability of such processes, the cost of cellulase production and enzymatic hydrolysis should be reduced. For this, recycling cellulase can be considered for reducing the saccharification cost of lignocellulose. In this study, cellulase recycling for high-solids enzymatic hydrolysis (i.e., 20%) was evaluated in saccharification of hydrothermally-pretreated EFBs. Results High-solids (20%) enzymatic hydrolysis of hydrothermally-pretreated empty fruit bunches with 40 FPU of Cellic CTec3/g glucan was carried out for cellulase recycling. In the second round of hydrolysis using a recycled enzyme, only 19.3% of glucose yield was obtained. The most important limiting factors for cellulase recycling of this study were identified as the enzyme inhibition by glucose, the loss of enzyme activities, and the non-productive binding of enzymes to insoluble biomass solids. To overcome these limitations, PEG was added prior to the first-round hydrolysis to reduce non-productive enzyme binding, glucose was removed from the enzyme fraction to be reused in the second-round hydrolysis, and EFB solids from the first-round hydrolysis were used in the second-round hydrolysis. These three additional measures with cellulase recycling resulted in a 3.5 times higher glucose yield (i.e., 68.0%) at the second round than that of the control, the second-round hydrolysis with cellulase recycling but without these measures. Conclusions Because of the high obstacles found in this study in achieving high saccharification yields in the high-solids saccharification of high-lignin lignocellulose with cellulase recycling, effective measures for improving enzymatic saccharification yields need to be accompanied with cellulase recycling.

Background
An increasing demand of fossil fuels and the recent environmental issues have promoted the development of bioproducts from renewable resources [1,2]. Among renewable resources, lignocellulosic biomass feedstocks such as empty fruit bunches (EFBs) are attractive due to their abundances, low prices, and high carbohydrate contents [3]. The typical conversion process of lignocellulosic biomass into chemicals, fuels, and materials mainly includes pretreatment, enzymatic hydrolysis, fermentation, and recovery of final products [4]. To increase the feasibility of commercializing such processes on a large scale, it is necessary to improve the process economics, and the cost reduction in cellulase production and enzymatic hydrolysis can be one example [5,6].
Several approaches have been adopted to reduce cellulase costs, including protein engineering of cellulase [7], optimization of cellulase formulations [8], addition of accessory agents to cellulase such as synergistic proteins [9] and surfactants [10], and cellulase recycling [11]. Among these, cellulase recycling is a simpler and more practical approach that reuses the cellulase recovered from the hydrolysate from the preceding process into fresh biomass. To date, significant reduction in cellulase usage with glucose yields of 60-80% has been demonstrated on a laboratory scale via cellulase recycling: for example, four consecutive recyclings of 20 filter paper units (FPU) of Celluclast 1.5 L/g glucan for 2% (w/v) loading organosolv pretreated lodgepole pine, thus resulting in saving 80 FPU enzyme but losing 9.1% glucose yield [12]; three consecutive recyclings of 15 FPU of Spezyme CP/g glucan at 5% (w/v) for ammonia pretreated corn stover, thus resulting in saving 45 FPU enzyme but losing 26.8% glucose yield [13]; and three consecutive recyclings of 20 FPU of fungal cellulase/g glucan for 2% alkali pretreated wheat straw, thus resulting in saving 60 FPU enzyme but losing 12.8% glucose yield [14]. Nevertheless, such studies on cellulase recycling were performed in low-solids enzymatic hydrolysis (i.e., < 15%, w/v). In addition, these studies commonly indicated that solids containing lower lignin (i.e., < 10%, w/w) were suitable for cellulase recycling [12,14].
In this study, cellulase recycling for high-solids enzymatic hydrolysis (i.e., 20%) was evaluated through the saccharification of hydrothermally-pretreated EFBs containing 30.8% of lignin. First, the effects of cellulase and EFBs loadings on the glucose yield were tested for highsolids enzymatic hydrolysis of pretreated EFBs. Next, the highly relevant limiting factors hindering cellulase recycling under the present experimental conditions were identified. Eventually, additional process-oriented measures were developed to overcome the limitations of cellulase recycling, thereby increasing the efficiency of cellulase recycling for high-solids enzymatic hydrolysis.
After hydrothermal pretreatment, the recovery yield of insoluble solids was 67.0% (w/w) of initially loaded solids, and the glucan, hemicellulose, and lignin content (w/w) were 54.7%, 12.0%, and 30.8%, respectively, in the insoluble solids of pretreated EFBs (Table 1). Thus, the recovery yields (w/w) of the biomass components, glucan, hemicellulose, and lignin were determined as 98.0%, 33.6%, and 92.7%, respectively. The loss of glucan after the pretreatment was negligible at the present hydrothermal pretreatment at 190 °C for 15 min. The recovery yield of lignin was considerably high (92.7%). In contrast, a significant loss of hemicellulose (66.4%) was observed after the pretreatment. The substantial loss of hemicellulose and the insignificant losses of cellulose and lignin, observed in this study, were considered to follow the typical characteristics of the hydrothermal pretreatment.

Effects of cellulase and EFBs loadings on the glucose yield
To investigate the effects of cellulase and EFB solids loadings on the glucose yields from the enzymatic saccharification of hydrothermally-pretreated EFBs, enzymatic hydrolysis was performed at 50 °C for 72 h by varying the loadings of EFB solids and cellulase (i.e., Cellic CTec3). In particular, the loading ranges of EFB solids and cellulase were 20-30% (w/v) and 10-60 FPU/g glucan, respectively. As a result, the glucose yield increased with increasing cellulase loading, but decreased with increasing solids loading (Fig. 1). The decrease in glucose yield with increasing a solids loading could be due to mass transfer limitation as previously reported [16].
At the 30% EFB solids loading (Fig. 1a), the liquefaction of solids slurry was slower in the initial period of enzymatic hydrolysis (e.g., 24 h) than at other EFB solids loadings. For instance, the liquid fraction of hydrolysates obtained with 10 FPU of Cellic CTec3/g glucan could not be collected even after 36 h (Fig. 1a). At higher solids loadings, mass transfer and mixing became limited due to the lack of free water, resulting in retarding the liquefaction of solids slurry [16]. Liquefaction is one of the key factors for efficient high-solids enzymatic hydrolysis, as it can affect the rheological behaviors between the biomass solids and endoglucanase [17]. Meanwhile, at the 30% EFB solids loading, the highest glucose yield, 91.7%, was obtained at a cellulase loading of 60 FPU/g glucan after 72 h. At the 25% EFB solids loading (Fig. 1b), liquefaction was faster than that at the 30% solids loading, and 79.3%, 98.1%, and 100.0% of glucose yields were obtained after 72 h at cellulase loadings of 20, 40, and 60 FPU/g glucan, respectively. At the 20% solids loading (Fig. 1c), 91.8% and 100.1% of glucose yields were obtained only after 48 h at cellulase loadings of 40 and 60 FPU/g glucan, respectively. Among the cellulase loadings tested in Fig. 1, 10 FPU/g glucan is known as the most economicallyfeasible cellulase loading [18]. However, in this study, with 10 FPU/g glucan, only 56.1%, 62.2%, and 67.3% of glucose yields were obtained at 30%, 25%, and 20% solids loadings, respectively (Fig. 1), and these glucose yields were too low to readily observe the effect of cellulase recycling at high-solids loadings. Furthermore, with 20 FPU/g glucan, the glucose yield and volume of the liquid fraction separated for cellulase recycling were much lower than those with 40 FPU/g glucan at 48 h. Thus, 40 FPU and 60 FPU of Cellic CTec3/g glucan as the cellulase loadings, resulting in high glucose yield and titer, were selected to evaluate the feasibility of cellulase recycling at high-solids enzymatic hydrolysis of pretreated EFBs. In addition, based on the rate of enzymatic hydrolysis (Fig. 1a, b), 20% of EFB solids loading and 48 h time point, at which the glucose yield was saturated, were selected as the conditions for evaluating cellulase recycling. Under these saccharification conditions, 108.5 g/L and 118.4 g/L of glucose can be produced from 20% solids with 40 FPU and 60 FPU of Cellic CTec3/g glucan for 48 h, respectively.

Recycling cellulase
To test the feasibility of cellulase recycling in the enzymatic hydrolysis of hydrothermally-pretreated EFBs, the liquid fractions of EFBs hydrolysates were collected after 48 h of the first-round enzymatic hydrolysis and were recycled further (i.e., the second and third rounds) for the enzymatic hydrolysis of pretreated EFBs. After the first-round enzymatic hydrolysis for 48 h, 96.7% and 98.3% of glucose yields were obtained with the glucose titers of 119.3 g/L and 123.6 g/L at the cellulase loadings of 40 FPU and 60 FPU/g glucan, respectively ( Fig. 2a, b). At the end of the first-round enzymatic hydrolysis, the hydrolysate was filtered and 11.6 ± 0.6 mL of filtrate was obtained, which was 72.5% of the initial liquid volume of the hydrolysis reaction mixture. For the second-round enzymatic hydrolysis, the filtered liquid fraction was applied to the fresh pretreated EFBs. As a result, 19.3% and 20.5% of glucose yields were obtained during the second round of enzymatic hydrolysis at the cellulase loadings of 40 and 60 FPU/g glucan, respectively (Fig. 2b). For the third round of enzymatic hydrolysis, only 7.9 ± 0.4 mL of the liquid fraction filtrate was obtained due to the liquefaction in the second-round enzymatic hydrolysis. After the third-round enzymatic hydrolysis, glucose yields of less than 5% were obtained (Fig. 2b).
In this study, the increase in cellulase loading from 40 to 60 FPU/g glucan did not increase the efficiency of cellulase recycling, suggesting the existence of possible obstacles for cellulase recycling in the high-solids enzymatic hydrolysis, particularly the high lignin contents in biomass. Even at a low-solids enzymatic hydrolysis, cellulase recycling was hampered by the high lignin contents of biomass (i.e., 20-32%, w/w): 100% of glucose yield of steam-exploded loblolly pine at the first-round hydrolysis with 20 FPU Spezyme, 60% at the second round, and 15% at the third round [12]; 68% of glucose yield of acidpretreated wheat straw (2%, w/w) at the first round with 20 FPU cellulase, 44% at the second round, and 27% at the third round [14]. Thus, the strategy of alleviating the binding of cellulase and lignin was suggested to overcome the limitations in the cellulase recycling [19].

Limiting factors in cellulase recycling during high-solids enzymatic hydrolysis Product inhibition of enzymes by glucose
Although high-solids enzymatic hydrolysis can produce high titer glucose, residual glucose during the enzymatic hydrolysis significantly limits the yield of enzymatic hydrolysis [20]. Considering the concentration of glucose to be as high as 85.0 g/L in the EFBs hydrolysates during commencement of the second-round enzymatic hydrolysis (Fig. 2a), such a high titer may seem to be a limiting factor for the high-solids enzymatic hydrolysis. Although the inhibition of cellulase by glucose is well known, the degree of glucose inhibition can be significantly affected by the types and concentrations of substrates and enzymes used [21]. Thus, to investigate whether the residual glucose is the limiting factor under the present experimental conditions, glucose was added at various amounts to the enzymatic hydrolysis of pretreated EFBs. As a result, as the residual glucose concentration increased from 0 to 108.0 g/L, the glucose yield decreased from 100.0 to 60.9% (Fig. 3a). This decreased glucose yield may be due to the product inhibition to cellulase by glucose [22]. Therefore, prior to the second-round enzymatic hydrolysis, 85.0 g/L, the initial glucose concentration, which was carried over from the filtrate of the liquid fraction of the first-round hydrolysate, needs to be reduced.

Loss of residual enzyme activity in the liquid fraction after enzymatic hydrolysis
At the typical enzymatic hydrolysis conditions for lignocellulose, pH 4.8 and temperature of 50 °C [23], β-glucosidase originating from Trichoderma reesei in Cellic CTec2 was found to bind onto lignin, thus resulting in a significant activity loss of residual β-glucosidase in the liquid fraction [24]. This loss of activity can lead to accumulation of cellobiose with producing less glucose [25]. In this study, the activity loss of β-glucosidase in Cellic CTec3 was hypothesized as one of the most relevant limiting factors in cellulase recycling, and the β-glucosidase activity was monitored in the first-round enzymatic hydrolysis of pretreated EFBs. In addition, the activities of total cellulase and CMCase (carboxymethyl cellulase) were also monitored. In particular, the endoglucanase, whose activity is expressed by CMCase, plays a key role in the liquefaction of high-solids enzymatic hydrolysis [17].
In this study, after the first-round enzymatic hydrolysis for 24 h, all of the three enzyme activities, namely, total cellulase, endoglucanase, and β-glucosidase, were retained less than 20% of their initial activities (Fig. 3b). Thus, it was supposed that productive binding of enzymes onto cellulose was dominant for 24 h, while non-productive binding of enzymes onto lignin was dominant after 48 h. In particular, β-glucosidase retained less than 1.1% of its initial activity after 24 h For cellulase recycling at the next-round enzymatic hydrolysis, the liquid fraction obtained from preceding rounds of enzymatic hydrolysis was directly applied to fresh pretreated EFBs with sodium citrate buffer (50 mM, pH 4.8). Experimental data are expressed as mean ± standard deviation from three independent experiments of enzymatic hydrolysis. Similar severe activity loss of β-glucosidase was reported in Cellic CTec2 when incubated with stream-pretreated EFBs comprising a high lignin content (e.g., 37.6%) [26], presumably due to the non-productive binding of β-glucosidase to lignin [24]. With regard to the total cellulase and CMCase activities, 5.4% and 15.0% were retained after the first-round enzymatic hydrolysis at 48 h (Fig. 3b). The higher loss of the total cellulase activity, which was measured by the glucose production from filter paper, than the endoglucanase activity may be due to the significant activity loss of β-glucosidase that primarily produces glucose from cellobiose. Accordingly, at the end of the first round of hydrolysis, cellobiose was not detected because the glucose yield reached almost 100%. However, at the end of the second round of hydrolysis, cellobiose accumulated up to 21.4 g/L (data not shown).

Binding of enzyme proteins to insoluble biomass solids
As observed in the non-productive binding of enzymes (e.g., β-glucosidase) to lignin [24], the significant losses of remaining enzyme activity in the liquid fraction in this study after enzymatic hydrolysis was hypothesized to be mainly due to non-productive binding of enzymes to the insoluble solids of pretreated EFBs including lignin [27]. To validate this hypothesis, protein adsorption during enzymatic hydrolysis was monitored (Fig. 3c). As a result, only 16.7% of the initial amount of proteins were recovered after 24 h of enzymatic hydrolysis. After the completion of enzymatic hydrolysis at 48 h, the amount of proteins recovered from the liquid fraction were still as low as 37% of its initial amount (Fig. 3c). These results indicated that most of enzyme proteins of Cellic CTec3 still remained in the solid fraction after enzymatic hydrolysis. Typically, enzymes bind onto solids immediately in the beginning of hydrolysis. Once enzymatic hydrolysis occurs, enzymes bound to solids start being detached from the solids [28]. However, approximately 40% of the initial enzymes still remain in the solids after the completion of enzymatic hydrolysis even in low-solids enzymatic hydrolysis [12,28]. Considering that most of cellulose and hemicellulose were hydrolyzed after 48 h in this study, enzymes may have been adsorbed onto lignin. Thus, it was supposed that productive binding of cellulases onto cellulose was dominant during enzymatic hydrolysis according to the residual activity of cellulases in the liquid fraction. However, it was supposed that non-productive binding of cellulases onto lignin was dominant after the completion of enzymatic hydrolysis due to the depletion of cellulose.

Effect of glucose separation on cellulase recycling
To alleviate the product inhibition of enzymes by glucose, as observed earlier in this study, it is necessary to remove glucose from the liquid fraction of EFBs hydrolysates between the first-and second-round enzymatic hydrolysis. To accomplish this, the glucose and enzymes in the liquid fraction from the first-round enzymatic hydrolysis were separated using 10 kDa ultrafiltration (UF) membrane, and the UF filtrate containing glucose was separated from the cellulase-containing fraction. When switching from the liquid fraction without using UF to the UF-separated enzyme fraction for the second-round enzymatic hydrolysis, the glucose yield increased by 1.9 times, from 19.3 to 36.3% (Fig. 4).

Effect of polyethylene glycol on cellulase recycling
To alleviate the significant loss of β-glucosidase activity, presumably due to its non-productive binding of β-glucosidase onto lignin, as observed in this and other studies [24,27], 62.5 mg of polyethylene glycol (PEG) 8000/g glucan was added prior to the first round of enzymatic hydrolysis of pretreated EFBs, as previously described [29]. In this study, when PEG was added, the glucose yield from the second-round enzymatic hydrolysis was 60.4%, which was 3.1 times higher than that of the untreated control (i.e., 19.3%; Fig. 4) and which was 1.6 times higher than that obtained after glucose separation. Therefore, not only the PEG addition but also the glucose separation was found to be effective in increasing the enzymatic hydrolysis of pretreated EFBs using recycled cellulases. This effect of PEG on the enzymatic hydrolysis was previously suggested to be due to the reduction of non-productive binding of cellulases onto insoluble solids of pretreated lignocellulose [29,30].

Effect of used biomass solids with bound enzymes on cellulase recycling
So far in this study, to overcome the limiting factors of cellulase recycling, the separation of glucose and the addition of PEG were additionally performed and resulted in a 3.1 times yield increase in the second round of enzymatic hydrolysis compared to the glucose yield of the second round of cellulase recycling without these two measures. To furthermore increase the reusability of enzymes bound to the residual solids in the first-round enzymatic hydrolysis, 33.0% of the used solids from the first-round hydrolysis were directly applied to the second-round enzymatic hydrolysis. Meanwhile, when performing the enzymatic hydrolysis with more than 33.3% of used solids (2.6 g of 7.9 g), the liquefaction was rarely observed due to high viscosity at a high solids loading. After the second-round enzymatic hydrolysis, a glucose yield of 68.0% was obtained, which was 7.6% higher than that obtained after glucose separation and PEG addition, but without the reuse of the used solid fraction (Fig. 4).
Overall, when introducing the additional three measures, glucose separation, PEG addition, and the reuse of the used solids fraction into the second-round enzymatic hydrolysis, the glucose yield increased by 3.5 times compared to that of control without these efforts. Using the above three additional measures for cellulase recycle, when the three scenarios of enzymatic hydrolysis of pretreated EFBs, the two separate hydrolysis using 20 FPU of Cellic CTec3/g glucan, and the consecutive hydrolysis through cellulase recycling using 40 FPU/g glucan with or without PEG and used biomass solids, were compared for their overall glucose yield from the two rounds ( Table 2). The overall glucose yield with cellulase recycling without the three measures was rather lower than that of the separate hydrolysis without cellulase recycle ( Table 2) due to the low glucose yield in the second round. However, the cellulase recycling with the three additional measures showed a 6.6% higher glucose yield than that from the separate hydrolysis. These results

Control
Glucose removed  . 4 Effects of the glucose removal from the enzyme fraction to be recycled, the addition of PEG to reduce enzyme binding, and the use of biomass solids from the first-round enzymatic hydrolysis, on the glucose yield of the second round in the two-round enzymatic hydrolysis of hydrothermally-pretreated EFBs with cellulase recycling Table 2 Effect of cellulase recycling on the overall glucose yields and total amounts of produced glucose (mean ± standard deviation) from the enzymatic hydrolysis of hydrothermally-pretreated EFBs a The first round of enzymatic hydrolysis b The second round of enzymatic hydrolysis c The overall glucose produced from 4 g of pretreated EFBs at the first round and 4 g of pretreated EFBs at the second round imply that the cellulase recycling itself cannot benefit the overall glucose yield without additional measures for improving hydrolysis efficiency. Also, to further increase the glucose yield from the aspect of enzymes, the protein engineering and formulation of enzymes can be performed to reduce electrostatic interactions between enzymes and lignin and to achieve a more favorable distribution of enzymes during enzymatic hydrolysis.

Conclusions
Cellulase recycling for high-solids enzymatic hydrolysis is important and challenging, especially, when pretreated biomass contains a high lignin content. In this study, it was found that cellulase recycling in the enzymatic hydrolysis of hydrothermally-pretreated EFBs (20%, w/v) was hampered by product inhibition of enzymes by glucose, activity losses of enzymes after hydrolysis, and binding of enzymes to insoluble solids of biomass.
To overcome these obstacles for cellulase recycling, glucose was removed from the enzyme fraction to be reused, PEG was added to reduce enzyme binding, and used EFB solids from the first-round hydrolysis were added to reuse enzymes bound to the solids. As a result, a 3.5 times higher glucose yield than that of untreated control was obtained.

Hydrothermal pretreatment of EFBs
The EFBs used in this study were provided by the Korindo Group (Jakarta, Indonesia). These EFBs were ground and sieved to obtain approximately 850 μm particle sizes, which were then subjected to hydrothermal pretreatment at 190 °C for 15 min at a solid-to-liquid ratio of 1:11.5 (w/v). Briefly, 800 g of dry weight EFBs were soaked in 16 L of deionized water at 50 °C for 16 h, and were dewatered by centrifugation. Thereafter, they were transferred into a 15 L pretreatment reactor (Hanwoul Engineering, Gunpo, Korea) containing 9.2 L of deionized water. The temperature of the pretreatment reactor was increased to 190 °C with a 50 min ramping time and was maintained at 190 °C for 15 min to pretreat the EFBs, which were then discharged into a fabric bag with approximately 6 μm diameter pores and washed with water. Eventually, the pretreated EFBs were dried in a freeze dryer (IlShinBioBase, Dongducheon, Korea) and stored at − 20 °C until further use.

Compositional analysis of unpretreated and pretreated EFBs
Compositions of unpretreated and pretreated EFBs were analyzed according to the Laboratory Analytical Procedure (LAP) of National Renewable Energy Laboratory (NREL; Golden, CO) [31][32][33]. After the two-step acid hydrolysis, the sugar contents in the liquid fraction were measured using high-performance liquid chromatography (HPLC; Agilent 1100, Agilent Technologies, Waldronn, Germany) equipped with Aminex HPX-87H column (Bio-Rad, Hercules, CA). The acid-soluble lignin content in the liquid fraction was measured at 205 nm using a spectrophotometer (xMark, Bio-Rad, Hercules, CA). For determining the acid-insoluble lignin content, the liquid fraction was filtered and incubated in a furnace at 575 °C for 3 h, and the acid-insoluble lignin was weighed and expressed as the percentage (w/w) of the total dry weight of insoluble solids of biomass. To determine the ash content, EFBs were incubated at 575 °C for 24 h, and the weight of the residual ash was expressed as the percentage of the total biomass [31]. The recovery yield (%) of each component of insoluble biomass after pretreatment was determined by comparing the total dry weight of recovered solids and biomass composition before and after pretreatment (Table 1).

Enzymatic hydrolysis and cellulase recycling
Enzymatic hydrolysis was performed in duplicate according to the LAP of NREL [23]. Briefly, for each enzymatic hydrolysis, pretreated EFBs with dry wt. of 4 g were transferred into a glass vial containing 20 mL of the total reaction mixture comprising sodium citrate buffer (50 mM, pH 4.8), 0.02% (w/v) of sodium azide, and 10-60 FPU of cellulase/g glucan. Thereafter, the mixture was incubated at 50 °C with shaking at 200 rpm for 72 h. To examine cellulase inhibition by its product, enzymatic hydrolysis was performed in the presence of glucose at different concentrations, 6.3, 12.5, 25, 50, and 100 g/L. Glucose released during the enzymatic hydrolysis was measured using the HPLC, and the glucose yield (%) was expressed as the percentage of the theoretical maximum glucose to be released from the total amount of glucan in input pretreated EFBs. Cellulase used in this study was Cellic CTec3, which was provided by Novozymes Korea (Seoul, Korea), and the filter paper unit of Cellic CTec3 was measured at 191.6 FPU/mL. For cellulase recycling, following enzymatic hydrolysis for 48 h, the insoluble solids and the liquid fraction of the hydrolysates were separated by filtration using a 0.2 μm polyethersulfone membrane filter (Thermo Fisher Scientific, Waltham, MA). The collected liquid fraction was mixed with 4 g of fresh EFBs and sodium citrate buffer (50 mM, pH 4.8), resulting in a 20% (w/v) solids loading in a 20 mL reaction mixture. Thereafter, the reaction mixture was incubated at 50 °C with shaking at 200 rpm for 48 h, and the glucose yield (%) was determined as described above. To determine the glucose released during the second-or third-round enzymatic hydrolysis, the carried glucose from the previous round was subtracted.

Analysis of residual enzyme activity and protein concentration
After enzymatic hydrolysis for 48 h, the residual sugar from the liquid fraction of hydrolysate was completely removed using a dialysis membrane with a molecular cut-off of 10 kDa (SnakeSkin, Thermo Fisher Scientific, Waltham, MA) in 5 L of sodium citrate buffer (50 mM, pH 4.8) at 4 °C for 4 h. Thereafter, the enzymatic activity and protein concentration of the liquid fraction were measured.
The total cellulase activity in an enzymatic reaction mixture expressed as FPU/mL was determined according to the LAP of NREL [34]. Briefly, 0.5 mL of a diluted sample containing enzymes was incubated with 50 mg of filter paper strip (Whatman No. 1, Whatman, Maidstone, UK) and 1.0 mL of sodium citrate buffer at 50 °C for 60 min. The enzymatic reaction was terminated by adding 3.0 mL of a 3,5-dinitrosalicylic acid (DNS) agent at 95 °C for 5 min. Thereafter, the amount of released glucose was measured as reducing sugar at 540 nm using a microplate spectrophotometer (xMark). One unit of cellulase (FPU/mL) was defined as the amount of enzyme releasing 2.0 mg of glucose for 60 min.
The residual carboxymethyl cellulase (CMCase) activity in an enzymatic reaction mixture was determined by incubating 0.5 mL of a diluted sample containing enzymes with 0.5 mL of 2% (w/v) carboxymethyl cellulose (CMC; Sigma-Aldrich, St. Louis, MO) at 50 °C for 30 min [35]. The enzymatic reaction was terminated by adding a DNS agent, and the amount of released glucose was measured as reducing sugar at 540 nm. One unit of CMCase was defined as the amount of enzyme releasing 0.5 mg of glucose for 30 min.
The residual β-glucosidase (BG) activity in an enzymatic reaction mixture was determined, as previously described [36]. Briefly, 0.5 mL of a diluted sample containing enzymes was incubated with 1.0 mL of 2 mM of p-nitrophenyl-β-d-glucopyranoside (pNPG; Carl Roth, Karlsruhe, Germany) at 50 °C for 10 min. The enzymatic reaction was terminated by adding 2.0 mL of 1 M sodium carbonate. The amount of p-nitrophenol released from pNPG during the enzymatic reaction was measured at 410 nm. One enzyme unit of β-glucosidase was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per min. The protein concentration was measured using a bicinchoninic acid protein kit (Thermo Scientific, Rockford, IL).


extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Effects of Dietary Rumen-Protected Betaine Supplementation on Performance of Postpartum Dairy Cows and Immunity of Newborn Calves

Simple Summary Betaine plays an important role in growth, lactation, protein synthesis, and fat metabolism in animals, but there are few studies on transition dairy cows and newborn calves. The aim of the current study was to evaluate the effects of rumen-protected betaine supplementation from four weeks before expected calving to six weeks postpartum regarding the lactation performance and blood metabolites of dairy cows and immunity of newborn calves. The results suggested that betaine supplementation tended to increase fat mobilization of postpartum dairy cows. Furthermore, compared to the control calves, the betaine calves had greater plasma total protein and globulin concentrations, which indicates that the immunity of the betaine calves might have improved. Abstract The objective of this study was to evaluate the effects of rumen-protected betaine supplementation on performance of postpartum dairy cows and immunity of newborn calves. Twenty-four multiparous Holstein dairy cows were randomly divided into the control (CON, n = 12) and rumen-protected betaine (BET, n = 12) groups after blocking by parity and milk yield during the previous lactation cycle. The cows were fed a basal total mixed ration diet without BET (CON) or with BET at 20 g/d per cow (BET) from four weeks before expected calving to six weeks postpartum. The results showed that betaine supplementation had no effect on dry matter intake and milk yield of the cows. The BET cows tended to increase feed efficiency (energy-corrected milk/dry matter intake) and body weight loss postpartum compared to the CON cows. The plasma β-hydroxybutyrate concentrations of the BET cows were greater at d seven after calving than those of the CON cows. Moreover, compared to the CON calves, the BET calves had greater plasma total protein and globulin concentrations. The plasma glucose concentrations of the BET calves tended to decrease relative to CON cows. In conclusion, rumen-protected betaine supplementation from four weeks before expected calving tended to increase fat mobilization of postpartum dairy cows, and might improve the immunity of newborn calves.

Introduction
During the transition period, dairy cows are in a state of great metabolic stress because of the increased demand for nutrients to maintain fetal growth and milk synthesis. Transition dairy cows tend to have negative energy and amino acid balance after calving, which leads to an increase in fat and protein mobilization in tissues [1,2]. A negative methyl donor balance also likely occurs in transition cows because milk is high in methylated compounds [3]. Moreover, the last two months of gestation, where 60% of the body weight gain before birth occurs [4], is critical for bovine fetal development.
Betaine functions as a methyl donor and an organic osmolyte [2,5], which plays an important role in growth, lactation, protein synthesis, and fat metabolism in animals [6]. Betaine supplementation in the diets of steers increased body weight gain and fat deposition [7]. In lactating dairy cows, feeding betaine improved the milk yield and milk protein [8,9]. Supplementing betaine reduced plasma concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) of lactating dairy cows [10], but elevated the concentrations of NEFA and BHB of transition dairy cows to change lipid metabolism [11]. Furthermore, betaine is vital for fetal development [12], and is related to the offspring's weight and immunity [13]. However, due to the fast rumen degradation (approximately 45%/h) of betaine in vivo [14], unprotected betaine cannot be absorbed efficiently. Our previous study showed that dietary rumen-protected betaine supplementation in lactating dairy cows improved lactation performance and fat metabolism [15]. Whether it improves the performance of postpartum dairy cows and the immunity of newborn calves remains unexplored. Therefore, the objectives of the current study were to evaluate the effects of rumen-protected betaine supplementation from four weeks before expected calving to six weeks postpartum on the lactation performance and blood metabolites of dairy cows and immunity of newborn calves.

Animals and Treatments
All the experimental protocols used in this study were approved by the Animal Care Committee of Zhejiang University (Hangzhou, China) (Approval Number: ZJU20160379). Twenty-four multiparous prepartum Holstein dairy cows were selected and divided randomly into the control (CON, n = 12) and rumen-protected betaine (BET, n = 12) groups after blocking by parity (2.27, SD = 1.4) and milk yield during the previous lactation cycle (24.9 kg/d, SD = 6.0). The cows were fed a basal total mixed ration (TMR) diet (Table 1) without BET (CON) or with BET at 20 g/d per cow (BET), according to Zhang et al. (2014) [9] from four weeks before expected calving to six weeks postpartum. The basal diets were formulated based on the NRC (2001) [16]. The cows were fed three times daily at approximately 06:30, 13:30, and 19:30 h, and BET (BET with 30% purity, Hangzhou King Technology Feed Co., Ltd, Hangzhou, China) was supplemented twice daily in the morning and evening by top-dressing the TMR during feeding. All cows were housed in tie-stall barns and given access to fresh water ad libitum. After parturition, the cows were milked three times daily at approximately 07:00, 14:00, and 20:00 h. Fourteen calves randomly selected (CON calves: n = 7, BET calves: n = 7) were studied from birth to 24 h. All calves were weighed with a digital scale immediately after birth and were fed fresh colostrum from their dams within 2 h of birth. The calves were individually housed in hutches, and water was offered ad libitum.

Sample Preparation
The amounts of feed offered and refused were recorded according to Gu et al. (2018) [17] to determine dry matter intake (DMI). The TMR samples were collected weekly for dry matter (DM, 105 • C for 5 h), crude ash, ether extract (EE), crude protein (CP), and acid detergent fiber (ADF), according to AOAC procedures (method 942.05, 920.39, 988.05, and 973.18, respectively), and neutral detergent fiber (NDF) with sodium sulfite and amylase was analyzed [18]. Body weight (BW) was measured on d 0 and 42 after calving according to Wang et al. (2017) [19].
Milk yield was recorded for two consecutive days each week and milk samples from three consecutive milking were taken each week in the amounts proportional to the yield (4:3:3, composite from each daily milking). The samples were stored at 4 • C with bronopol tablets (D & F Control System Inc., San Ramon CA, USA) for later determination of protein, fat, lactose, total solids, and milk urea nitrogen (MUN) using a Combi Foss FT+ instrument (Foss Electric, Hillerød, Denmark). The 3.5% FCM (Fat-corrected milk) and ECM (Energy-corrected milk) were calculated by the formula [20] Blood samples from the cows were collected from the coccygeal vein in sodium-heparinized tubes at approximately 4 h after the morning feeding on −21, −10, 0, 7, 14, 28, and 42 d relative to calving. Blood samples from the calves were collected via the jugular vein using a sodium-heparinized tube shortly after birth before colostrum feeding and at approximately 24 h after birth. The samples were centrifuged for 10 min at 3000 g at 4 • C to harvest plasma, which was stored at -20 • C until analysis. The plasma samples were analyzed using an Auto Analyzer 7020 instrument (Hitachi High-Technologies Corp., Tokyo, Japan) with colorimetric commercial kits (Ningbo Medical System Biotechnology Co., Ltd., Ningbo, China) for total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), triglyceride (TG), cholesterol (CHOL), glucose (GLU), superoxide dismutase (SOD), NEFA, and BHB. The concentrations of globulin (GLOB) were calculated by the formula [21]: GLOB (g/L) = TP (g/L) − ALB (g/L).

Data Analysis
A randomized block design with repeated measures was used. The DMI, lactation performance, feed efficiency, and blood metabolites of the cows were analyzed with PROC MIXED of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Treatment, week, treatment × week, and block were included as the fixed effects in the model, and cow within treatment was used as a random effect. The blood metabolites of the calves were analyzed using the same procedure in SAS 9.2, except sampling hour instead of week was used as the repeated measure. The BW change of the cows, colostrum composition, and calves birth weight were analyzed using PROC MIXED of SAS 9.2 without the repeated statement. All associated interactions were removed from the model. The results are presented as least squares means. Statistical significance was determined at p ≤ 0.05 and tendencies at 0.05 < p ≤ 0.10.

Results and Discussion
Betaine supplementation had no effect on DMI, milk yield, and composition (p > 0.1, Table 2). Monteiro et al. (2017) [11] found that cows supplemented with betaine-containing molasses from 60 d before expected calving had higher milk yield, whereas no differences were observed in milk yield of cows supplemented with betaine-containing molasses from 24 d before expected calving, which is consistent with our results. The addition of betaine during the transition period increased the milk yield in a time-dependent manner, which might be related to the functions of betaine as an organic osmolyte to maintain the cell function by stabilizing cellular proteins and promoting proper protein folding [5,22]. The dry period is critical for the renewal and growth of mammary cells [23]. Hence, betaine addition during the far-off period has a positive effect on prepartum mammary growth, which increases the subsequent milk yield. The Lys: Met ratio in the postpartum diets was estimated to be 3.13:1 in our study, which had met the ideal Lys: Met ratio of 3.0:1 for an optimal milk protein content and yield [16,24]. Methyl donors (choline) additional supplementation had no detectable effect on cow performance when the Lys: Met ratio in diets had reached 3.0:1 [25]. This might also be a reason for BET additional supplementation, which has no effect on milk yield and composition. Compared to CON cows, BET cows tended to increase fat-corrected milk/dry matter intake (FCM/DMI, p = 0.09), energy-corrected milk/dry matter intake (ECM/DMI, p = 0.08), and BW loss postpartum (p = 0.10) ( Table 2). The plasma BHB concentrations of the BET cows were greater at d seven after calving than those of the CON cows (treatment × time: p = 0.07, Table 3). The greater number of animals in the study might have increased the statistical significance. The BET cows tended to have greater feed efficiency and BW loss postpartum in our study, coupled with greater concentrations of BHB at d seven after calving, which indicates that the BET cows might have an enhanced fat mobilization in early lactation due to higher milk yield numerically (milk yield was approximately 2.53 kg/d higher in BET cows than in CON cows) [11,26]. Table 2. Effects of supplementing cows without rumen-protected betaine (CON) or with rumen-protected betaine (BET) on dry matter intake, lactation performance, and body weight change during the first six weeks of lactation.  Table 3. Effects of supplementing cows without rumen-protected betaine (CON) or with rumen-protected betaine (BET) on blood metabolites from four weeks before expected calving to six weeks postpartum.  The plasma TP and GLOB concentrations of the BET calves were greater than those of the CON calves (p = 0.04, p = 0.05, respectively, Table 4), although no differences in calves birth weight were found between treatments (37.80 ± 1.68 kg vs. 36.03 ± 1.68 kg). The plasma TP and GLOB concentrations of calves increased significantly with maternal betaine supplementation, which indicates that it might improve the immunity of newborn calves because of maternal methyl donors supplementation [13,27]. Maternal dietary supplementation with methyl donors could program the health of offspring through the epigenetic regulation of the DNA molecule and cell signaling [27,28], which might improve the capacity for GLOB absorption of the intestine to improve the immunity of newborn calves. Furthermore, the lactose content of the BET colostrum tended to increase compared to the CON colostrum in our study (3.50% vs. 3.07%, p = 0.07), which might also have contributed to the results. Lactose plays a key role in the energy supply, absorption of minerals, and gastrointestinal functions of calves [29,30].

Items
The plasma GLU concentrations of the BET calves tended to decrease compared with those of the CON calves (p = 0.09, Table 4). The plasma SOD concentrations of the BET calves were greater at 2 h after birth than those of the CON cows (treatment × time: p = 0.01, Table 4). A positive correlation between neonatal glucose and cortisol concentrations proved that the lower concentrations of glucose in the BET calves were likely to have less stress around calving [31]. In turn, the degree of stress might influence newborn calves' energetic mobilization [31,32]. The greater plasma SOD concentrations 2 h after birth in our study suggested that the BET calves were in a state of less stress [33]. The plasma glucose concentrations in newborn calves might be related to the uteroplacental transport of glucose via mTOR signaling [34] and hepatic gluconeogenic gene expression via epigenetic mechanisms [35], which deserve further study.

Conclusions
Dietary rumen-protected betaine supplementation from four weeks before expected calving had no detectable effect on dry matter intake and milk yield, but tended to increase fat mobilization of postpartum dairy cows. Furthermore, the BET calves had greater plasma total protein and globulin concentrations, which indicates that the immunity of the BET calves might improve.


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Comparison between Organic and Conventional Spelt and Wheatgrass juice

This work is aimed to compare antioxidant and scavenger properties of conventionally and organically produced speltgrass. Moreover, the antioxidant status of spelt was compared with wheatgrass. Three spelt varieties were analyzed:Nirvana, Ebners–Rotkorn variety and Eco-10. Antioxidant enzymes, superoxide-dismutase and guiacol-peroxidase, were determined as well as ferric reducing antioxidant power and DPPH-antiradical power. Total soluble proteins and total phenols were determined and also lipid peroxidation as a parameter of oxidative stress. The organically produced spelt was exposed to the higher levels of oxidative stress than conventionally produced spelt, and also DPPH antiradical power was induced by conventional production, which is also demonstrated using the relative antioxidant capacity index. The best antioxidant properties, better than wheat, showed organically produced spelt genotype Ebners–Rotkorn which is determined by the highest phenol content, superoxide-dismutase activity, DPPH antiradical power and ferric reduced antioxidant power and consequently with the lowest lipid peroxidation intensity.

Introduction
Spelt (Triticum aestivum ssp. spelt) is an ancient subspecies of modern bread wheat (Triticum aestivum). For many years, cultivation of spelt declined, due to spelt's lower yield and its long straw with a tendency to lodge. In recent years, interest in the use of spelt increased, because it is considered as "healthier" and more "natural" grain than modern wheat. Spelt wheat is a suitable for growing without the use of pesticides, in harsh ecological conditions and in marginal areas of cultivation [1]. Moreover, spelt is more tolerant of poorly-drained and low-fertility soils than other commonly cultivated cereals [2].
Spelt possesses a valuable nutritional quality. Specificity of spelt in relation to wheat is in favor of spelt, which possesses more bioactive components in neo-saponified part of the liposoluble fraction [3]. It has higher protein content than wheat, some mineral contents, including selenium and zinc as well as high fiber content [4,5]. Wheat contains diverse bioactive compounds that may contribute to its antioxidant capacity. These bioactive components include carotenoids, tocopherols, tocotrienols, phenolic acids, phytic acid, phytosterols and flavonoids [6,7]. Previous studies have shown that wheat varieties vary in antioxidant properties as well as in composition in secondary biomolecules including antioxidants [8].
Wheatgrass (aboveground parts of the wheat) was traditionally used in ancient Egypt and perhaps even early Mesopotamian civilizations. Wheatgrass and spelt grass are powerful, concentrated liquid nutrients which have been used as home-made remedy or commercially distributed as frozen juice or powdered and freeze dried [9,10]. Like most plants, it contains chlorophyll, amino acids, minerals, vitamins, and enzymes. It was reported the significant presence of polyphenols and other flavonoids responsible for antioxidant activity as well as a diverse class of organic compounds in the aqueous extract, ranging from saturated and unsaturated fatty acids, alcohols and terpenes [11]. It has also been believed that wheatgrass could strengthen the immune system and increase the life span of cancer patients by regressing the spread of cancer cells [12].
Organic agriculture implies no use of synthetic fertilizers, genetically modified materials, sewage sludge or pesticides. Although the production and need for organic production are recently increasing in the world, it is necessary to find determination method for organic food quality determination with focus on laboratory methods [13]. Many studies found that organic methods produced lower yields and therefore required more land for a given production level [14]. A recent life cycle assessment (LCA) study showed that organic farming was either superior or similar to the conventional integrated production, but with exceptions for some products [15]. It was found that spelt endures well under suboptimal growing conditions [16], and to better utilize nutrients when grown in a low-input system [17], and show more resistance to a number of pathogens [18], than common wheat, thus spelt is recommended for organic agriculture.
Principal Component Analysis (PCA) was applied as a well known pattern recognition technique to the experimental data (used as descriptors) to characterize and differentiate among the observed samples.
Based on results obtained employing all antioxodant capacity (AOC) assays, relative antioxidant capacity index (RACI) has been calculated. Antioxidant efficiency of phenolics present in analyzed samples has been estimated based on phenolic antioxidant coefficients (PAC), determined as a ratio between particular AOC and total phenolic content (TPC), and compared to obtain a more comprehensive insight into the activity of analyzed samples.
In this way, the present study aimed to compare the antioxidant activity of organically and conventionally produced speltgrass and wheat grass. In order to evaluate antioxidant status of spelt and wheat antioxidant enzymes (superoxide-dismutase and guiacol-peroxidase), soluble proteins, total phenol content, 2,2´-Diphenyl-1-Picrylhydrazyl (DPPH) antiradical power, ferric reducing antioxidant power and lipid peroxidation were determined.

Material
The field experiment was carried out on three spelt variety (Triticum aestivum subs. spelt) and one wheat (Triticum aestivum). The spelt variety used in the experiment was Eco10 and Nirvana, which were supplied from a Serbian market and one variety (Ebners-Rotkorn) which was supplied from an Austrian market. The wheat variety used in the experiment was Europa 90.

Field experiment
The experiments were conducted at locality Institut "Tamiš" Pančevo (N 44° 56`, E 20° 43`) on the chernozem type of soil in the 2010-2011 vegetation season.The trial was set up in a randomized complete block design with four replicates. Each plot consisted of 10 rows, 10 cm apart and 5m long (the harvested area was 5m 2 ). Seedling density was considered as 500 seeds/m 2 . Growth stages of wheat were 30 (BBSH, 2001) [19].

Extraction procedures
Plant material was previously frozen at -70 °C in SNIJDERS deep freezer (VF360-86). For the freeze-drying process, frozen leaves were liophylizedin a Martin Crist Alpha 1-2 LDplus (Osterode, Germany) lyophilizator. The main drying process was performed at p = 0.12 mbar and temperatures from -40 ºC to 20 °C for 48 h. The final drying lasted 5 h at p = 0.005 mbar and temperatures from 20 ºC to 30 ºC. Plant material (1 g dry leaves) was extracted with 25 ml 80 % aqueous ethanol (0.1 M HCl) under 30 min sonication in an ultrasonic bath (Elmasonic S 100-H; Elma Schmidbauer GmbH) at ambient temperature. The extract was rapidly vacuum-filtered through a sintered glass funnel and kept refrigerated. This extract was used for determination of total phenol content, ferric reducing antioxidant power (FRAP) and DPPH antiradical power.
For the determination of lipid peroxidation (LP) and antioxidant enzymes, 2 g of plant material (fresh leaves) was extracted with 10 ml 0.1 M K 2 HPO 4 (pH 7.0) under 30 min sonication in an ultrasonic bath at ambient temperature. After10 minutes of centrifugation at 4 ºC and 10,000xg, aliquots of the supernatant were used for enzyme activity determinations. The used centrifuge was BOECO Germany (U-320-R).

Antioxidant enzymes and soluble proteins
The superoxide dismutase (SOD) activity was assayed according to Giannopolitis and Ries [20], by measuring the ability of the enzyme extract to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). Glass test tubes containing the mixture were immersed in a bath at 25 °C and illuminated by a fluorescent lamp (Philips MLL 5000W). Identical tubes, which were not illuminated, served as blanks. After illumination for 15 min, the absorbance was measured at 560 nm, using Thermoscientific UV-Visible spectrophotometer (Evolution 220). One unit of SOD was defined as the enzyme activity which inhibited the photoreduction of nitro-blue tetrazolium to the blue formazan by 50 %, and SOD activity of the extracts was expressed as SOD units per mg of protein. Reaction medium was prepared by mixing 2.6 mL 50 mM phosphate buffer (pH 7.8), 100 μL 13 mM methionine, 100 μL 75 μM NBT, 100 μL 0.1 mM EDTA and 50 μL 2 μM riboflavin.
The guaiacol peroxidase (GPx) activity was measured following the method of Kato and Shimizu [21]. The activity was calculated using the extinction coefficient of 26.6 mM -1 cm -1 at 470 nm for oxidized tetraguiacol polymer. One unit of GPx activity was defined as the calculated consumption of 1 μmol of H 2 O 2 min -1 mg protein -1 .
Soluble proteins were determined by the Bradford method (1976) [22]. Reaction medium contained 1 mL 0.07 MCommaise brilliant blue in 3 % HClO 4 , 1 mL destilled water and 25 μL of sample or standard (bovine serum albumin). After 5 min of incubation, absorbance of samples and standard was recorded at λ=595 nm. Protein concentration was read from calibration curve and finally expressed as mg protein/g dry matter.

Total phenols content (TPC)
The total phenolic content was determined by a modification of the Foline-Ciocalteu method and the results expressed as mg Gallic acid/100 g dry weight [23,24]. In 4.2 mL of distilled water, 50 µL of the corresponding extract was added and 250 µL of Folin&Ciocalteu-phenol reagent (previously diluted 1:10 with distilled water). The mixture was neutralized with 20 % sodium-carbonate solution after 5 minutes, and the content transferred into a 1.5 mL cuvette (1 cm path); absorbance was determined after 60 min at 720 nm.

DPPH antiradical power (DPPH)
The scavenging activity against DPPH radical was evaluated according to the method of Brand-Williams, Cuvelier, and Berset (1995) [25] with minor modifications. The assay mixture contained 2.9 mL DPPH solution (90 µmol/L) and 0.1 mL of the sample solution containing from 1 to 100 mg/mL dry extract; the final volume was 3 mL. The mixture was shaken vigorously on a Vortex mixer, then incubated 60 min at 25 °C in a water bath in the dark, after which the absorbance of the remaining DPPH was determined at 517 nm against a blank. Blank was sample at the same concentration above described containing all reagents except DPPH. For each sample three replicates were carried out. Radical Scavenging Capacity (RSC) was calculated by the Eq. (1): where A 0 is the control and A 1 is the sample solution absorbance. The concentration (in the final reaction medium in each method) that causes a decrease in the initial absorbance (control) by 50 % is defined as IC 50 . The IC 50 values for all RSC determinations were determined by polynomial fitting of the inhibition values using software ORIGIN 9.1. DPPH antiradical power defined as: (2)
The 100 μM Fe 2 + solution was used as a standard; 1 FRAP unit = 100 μM Fe 2 + . Total antioxidant capacity was expressed in FRAP units.

Lipid peroxidation (LP)
Lipid peroxidation was estimated based on thiobarbituric acid (TBA) reactivity. Samples were evaluated for malondialdehyde (MDA) production using a spectrophotometric assay for TBA. The extinction coefficient at 532 nm of 153,000 mol -1 cm -1 for the chromophore was used to calculate the MDA-like TBA produced. The color intensity of the MDA-TBA complex in the supernatant was measured by its absorbance at 532 nm [27].

Statistical analysis
All determinations were performed in quadruplicate. Statistical comparisons between samples were performed with Student's t-test for independent observations. Differences were considered significant at p < 0.05. The antioxidant test results were investigated with multivariate analysis. The correlation matrix was calculated, giving the correlation coefficients between each pair of variables, i.e. the analytical parameters tested. Each term of the matrix is a number ranging from -1 to +1: the + or -sign indicates a positive or negative interdependence between variables (direction), and the absolute value indicates the strength of the interdependence. Correlations between different parameters were considered significant at p < 0.05 level. Principal component analysis (PCA) was used to discover the possible correlations among measured parameters, while Cluster analysis (CA) is used to classify objects into groups.
The data were processed statistically using the software package STATISTICA 10.0 (StatSoft Inc., Tulsa, OK, USA).

Results and discussion
The widely used spectrophotometric assays, such as FRAP and DPPH, were applied in parallel with GPx, SOD and LP to measure AOC of antioxidant and scavenging activities of organic and conventional spelt and wheatgrass, which TPC was determined using FC assay was shown in Table 1. A large variation in phenolic content was noticed.
It was observed that conventionally produced Nirvana possessed 9.40 % higher soluble protein content than organically produced one. Between organically and conventionally produced Eco-10 no significant difference was observed. In both investigated spelt genotypes (Eco-10 and Nirvana), lipid peroxidation was higher in organically produced spelt for 11.95 % and 16.46 %, respectively.
Evidence suggests that organic cropping systems generally produce lower, more variable yields than systems employing synthetic fertilizers and chemical crop protection measures. According to Palmer et al. [28], the major limiting factor for yields in organic production systems is limiting nitrogen supply. Our results are in agreement with mentioned observations. Organic production of spelt did not provoke accumulation of soluble proteins, but contrary, the highest accumulation of soluble proteins was observed in conventionally produced spelt genotype Nirvana. Lipids are highly susceptible to oxidation and lipid peroxidation products, such as MDA quantity, are potential biomarkers for oxidative stress status in vivo [29]. Proteins are also the direct target for Reactive Oxygen Species (ROS) because of their high concentrations. Predictably, the lipid peroxidation intensity was significantly lower in conventionally produced spelt than in the organic spelt where protective measures did not apply. Values with the same letter are not statistically different at the p < 0.05 level (post hoc Tukey's HSD test); SPC (mg/ml) -soluble protein content; GPx (U/mg protein)-guaiacol peroxidase activity; SOD (U/mg protein) -superoxide dismutase activity; LP MDA (nmol/mg protein) () -lipid peroxidation enzyme, TPC (catechin eq (mg/100g dw))-total phenolic content

Soluble proteins and lipid peroxidation
The SOD activity ranged from 9.26 (wheat, organically produced) to 19.38 U/mg protein (Ebners-R spelt, organically produced). GPx activity ranged from 3.8 (Nirvana spelt, organic) to 9.05 U/mg protein (wheat, organically produced). No significant correlations between antioxidant enzymes and with LP and soluble proteins were found. Both antioxidant enzymes were significantly higher in organically produced spelt Nirvana, for 29.56 % (SOD) and for 83.16 % (GPx). Results are given in Table 1. SOD catalyzes the dismutation of superoxide radical in all living tissues, including plants, leading to hydrogen-peroxide production. Peroxidases are haem-containing enzymes that use hydrogen peroxide as the electron acceptor to catalyze a number of oxidative reactions. The conversion of superoxide into hydrogen peroxide and decomposition of hydrogen-peroxide presents the first line of cellular defense to prevent deleterious biological oxidation by oxygen radical generated during cellular metabolism. Despite the presence of an efficient antioxidant system, oxidative stress still occurs in plant cells, either due to uncontrolled production or inefficient scavenging of ROS [30]. The reaction of antioxidant system depends on a few crucial factors such as duration to which the cell gets exposed to the oxidative stress and ecological context of oxidative stress as well as the ratio between antioxidants and also between ROS (O 2 •-, H 2 O 2 , •OH). In fact, the antioxidant defenses in living cells can respond to prooxidative conditions, when oxidative stress is not very strong, with a compensatory mechanism that increases the antioxidant activities [31]. If oxidative stress is persisting, or its level is very high, the protein damage became profound and a decreased SOD activity may occur. Our results that antioxidant enzymes were induced in organic production are in agreement with results of Palmer et al. [28] who also indicated that environment drivers may affect biochemical indicators and yields in organic systems more than in conventional systems.
No significant differences between organic and conventional spelt were observed considering total phenol content and FRAP values. Nevertheless, organic produced spelt showed significantly lower DPPH than conventional, for 38.05 % (Eco-10) and 35.15 % (Nirvana). A significant positive correlation was found between total phenol content and FRAP values (r=0.861; p < 0.05). DPPH was also positively correlated, but insignificantly, with TPC and FRAP. Significant negative correlation was found between LP and DPPH (r= -0.869; p < 0.05). Correlation matrix is presented in Table 2.
According to Grinder-Pedersen et al. [32], different food production methods may result in differences in the content of secondary metabolites such as polyphenolic compounds. Polyphenolic compounds are strong natural antioxidants and also part of the plant defence system with variety of functions [33]. The presence and content of different polyphenols are intraspecifically dependant and also affected by cultivation, harvesting conditions such as growing conditions, degree of ripeness and variety of the plants. Only a limited number of studies have investigated the effect of cultivation technique on the content of flavonoids, and the results are inconsistent [32]. Chemicals used in conventional production such as synthetic herbicides, pesticides, insecticides and fertilizers may both decrease and increase the production of polyphenolic compounds in plants [34,35]. Organic food production is characterized by the absence or limited use of synthetic herbicides, pesticides, and insecticides and a lower use of fertilizers. Our results considering Eco-10 and Nirvana spelt genotypes pointed out no significant effect of the cultivation technique on total phenol content and FRAP values. These results are in accordance with previously reported studies of polyphenol content in three different strawberry cultivars in the organic and conventional systems where no difference was seen [35]. Nevertheless, DPPH was lower in conventionally produced spelt genotypes what can be explained by the direct effect of certain chemicals used in production or their metabolites on this DPPH radical scavenging, or by the accumulation specific polyphenols with high scavenging potential towards DPPH radical. The highest value for all three antioxidant parameters were detected for organic Ebners-R spelt variety.

Relative antioxidant capacity index (RACI) and phenolic antioxidant coefficients (PAC)
In various methods for AOC activity determination, efficiency of AOs can vary due to the different chemical mechanisms lying behind the assays. In order to achieve a more reliable comparison between the samples analyzed, two additional parameters such as a relative antioxidant capacity index (RACI), calculated by assigning equal weight to all AOC assays applied (including TPC as a measure of total reducing activity), and phenolic antioxidant coefficients (PAC), calculated as a ratio between particular AOC capacity and total phenolic content, were introduced. As seen in Fig. 1, the highest values of RACI were ascribed to spelt Ebners-R and wheat Europa 90 (0.501 and 0.447). Low but still positive values of RACI were obtained for organically grown spelt Nirvana (0.119) and spelt Eco-10 (0.057). The negative values of RACI were calculated for spelt Nirvana (-0.695) and spelt Eco-10 (-0.430), which were conventionally raised.
The PAC calculation enables the comparison of the effectiveness of phenolics present in analysed samples and also provides specific insight into differences between applied AOC assays. Variations of both PAC SOD and PAC FRAP were remarkable, while variations in PAC LP and PAC GPx were much lower. The lowest values of PAC DPPH have been ascribed to samples with higher values of RACI, i.e. high AOC value. High PAC of organic and conventional spelt and wheatgrass did not seem to be always associated with high AOC value (Fig. 2).

PCA analysis
The orientation of the vector describing the variable in factor space indicates an increasing trend of these variables, and the length of the vector is proportional to the square of the correlation values between the fitting value for the variable and the variable itself. The angles between corresponding variables indicate the degree of their correlations (small angles corresponding to high correlations).
The PCA of the presented AOC data explained that the first two components accounted for 74.32 % of the total variance (50.54 and 23.78 %, respectively) in the seven variables (AOC assays, TPC and SPC). Considering the map of the PCA performed on the data, LP (which contributed 23.1 % of total variance, based on correlations), exhibited a positive score according to the calculation of the first principal component, while SOD (13.8 %), TPC (19.0 %), FRAP (22.2 %) and DPPH (18.4 %) showed a negative influence for the first principal component (Fig. 3). The positive contribution to the second principal component was observed for SPC (21.1 % of total variance, based on correlations), while the negative contribution to the second principal component calculation was observed for GPx (34.7 %), SOD (13.4 %) and TPC (13.6 %). The points shown in the PCA graphics, which are geometrically close to each other indicate the similarity of patterns that represent these points. The first principal component is determined by the influence of LP, DPPH, FRAP, TPC and SOD, and it represents the differences in AOC of the samples. The most intensive AOC activity was observed for Ebners-R, while the lowest FRAP value was obtained for Eco-10. Nirvana and Europa samples were close to obtained AO activity. The second PC shows the differences in protein content. Conventionally grown samples were characterized by higher soluble protein content (Nirvana), while organically grown samples showed the higher GPx value.
The presented results represent a platform for the future research, which will be focused on the more detailed exploration of wheat and spelt grass phytochemical potential and corresponding biological activities.

Conclusion
Differences in antioxidant status of all three investigated Spelt genotypes (Eco-10, Nirvana and Ebners-Rotkorn) were observed between each other as well as in comparison with wheat. The highest superoxide-scavenging activity,  Table 1. ferric reducing antioxidant power, total phenol content, DPPH antiradical power and the lowest lipid peroxidation intensity was observed in Ebners-Rotkorn genotype, even better than organically produced wheat. Comparative analysis of organically and conventionally produced spelt genotypes Eco-10 and Nirvana indicated that DPPHantiradical power was induced in both genotypes by conventional production. Regardless the lower DPPH, and slightly increased LP, organically produced spelt possess high antioxidant capacity that can resist towards oxidative stress by induction of antioxidant enyzmes. The presented results represent a platform for the future research, which will be focused on the more detailed exploration of wheat and spelt grass phytochemical potential and corresponding biological activities.


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The value of biomarkers in colorectal cancer

Supplemental Digital Content is available in the text

Introduction
Colorectal cancer (CRC) is the third most common cancer in the world, with nearly 1.4 million new cases and 694,000 deaths each year. [1,2] It is the fourth most common cause of cancer death in the world and the second most common cause of cancer death in the Western world. [3][4][5] In many Asian countries, the incidence of CRC has increased 2 to 4 times over the past few decades. [6][7][8] Despite improvements in treatment strategies in recent years, the overall survival rate of CRC is still very low. [8] The survival rate of CRC depends to a large extent on the stage of the disease at the time of diagnosis. [9] The 5-year survival rate for CRC patients detected at the local stage is about 90% after curative surgery, 70% for regional patients, and approximately 10% for patients with disseminated disease. [9][10][11] Therefore, screening for rectal cancer at an early stage is essential. During the past several years, many different screening techniques have been used. However, these techniques are not ideal due to low specificity, low sensitivity, high invasiveness, or high cost. [12,13] Thus, new technologies are in urgent need for CRC early detection.
In recent years, an increasing number of studies has shown that biomarkers may have great potential for early screening of CRC and some biomarkers have been tested in systematic reviews JW and WJL are co-first authors.
The present study was supported by the Science Foundation of the First Hospital of Lanzhou University (No. ldyyyn2018-57), and the Postdoctoral Science Foundation (206706).
The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. (SRs). [14][15][16] As we know, well-conducted SRs and meta-analyses with high quality can provide the best evidence for clinical practice and healthcare decisions. [17][18][19] If these SRs did not carry out well, it will not only waste resources but also mislead clinical practice and even cause huge losses to human health and social property. However, no studies have been conducted to evaluate the quality of these SRs. Furthermore, these SRs did not clarify which biomarker is the optimal diagnostic test for early and accurate detection of CRC. This overview will evaluate the methodological quality of available SRs and compare the diagnostic value of different biomarkers in order to find the best biomarker for diagnosing CRC.

Methods
This protocol will be reported according to the preferred reporting items for systematic reviews and meta-analysis protocols (PRISMA-P) checklist. [20] As a part of our project, this protocol has been registered on the international prospective register of systematic review (PROSPERO) (CRD42019125880).
2.1. Selection criteria 2.1.1. Type of studies. We will include SRs that reported the diagnostic value of any biomarker in diagnosing CRC. Furthermore, the SRs must meet the participants, index tests, and outcomes clarified below.
2.1.2. Participants. CRC patients and the diagnosis of CRC was proven by histopathological analysis. There are no restrictions on age, race, sex, and nationality of participates, as well as treatment plan, stage and types of cancer.

Index tests.
The index tests can be a single biomarker or combined biomarkers as long as the index tests are used for detecting CRC. But combined index tests incorporate 1 biomarker and 1 imaging modality will be excluded.

Outcome measures.
SRs must explicitly state the sensitivity, specificity, diagnostic odds ratio (DOR) and their 95% confidence interval (95%CI) of biomarkers for each included original study. Or report the true positive, false positive, true negative, and false negative values for each original study to allow us to calculate the sensitivity and specificity.

Exclusion criteria.
(1) SRs did not perform the meta-analysis.
(2) SRs that did not report the diagnostic value of biomarkers which allow us to conduct quantitative analysis. (3) SRs did not include inclusion/exclusion criteria and adequate search strategy. (4) Conference abstracts, review articles, guidelines, consensus, documents or expert position papers, comments, letters, brief reports, proceedings, or protocol studies.

Literature search
The search strategies for relevant SRs were discussed by the review team and were established in co-operation with an experienced medical information specialist. [21] A comprehensive literature search for SRs published before February 2019 was conducted in the PubMed, Embase.com, Cochrane Library, and Web of Science without any language restrictions. The references of included SRs were also manually retrieved for additional relevant studies. The search strategy of the PubMed is presented in Supplementary 1, http://links.lww.com/MD/D34.

Selection of studies
The identified records were imported into EndNote X8 (Thomson Reuters (Scientific) LLC Philadelphia, PA) for management. After removing duplicated records, each study was screened by 2 reviewers independently based on titles and the abstracts. Any potentially eligible study was retrieved to obtain the full text for further evaluation. Then, all potential studies were evaluated independently by the same 2 authors according to the inclusion/exclusion criteria. Any disagreement was resolved by full discussion until a consensus was reached.

Data extraction and management
A predefined extraction form with detailed written instructions will be created using Microsoft Excel 2013 (Microsoft Corp, Redmond, WA, www.microsoft.com) to collect relevant information. Data extraction will be piloted on 10 studies to assess the reliability of data extraction across different reviewers. Then, 2 independent investigators will extract data from included SRs including general information, sample size, baseline characteristics, index tests, reference standard, and outcomes of the original study. The detailed information is presented in Table 1. If we identify multiple reviews addressing the same research question but share the same primary study, the data of the overlapping original studies will be included only once. If the same author reported their results acquired from the overlapping people or multiple published data in different original articles, only the most complete paper containing the most information is included. We will contact study authors for missing or unclear data. Disagreements on extractions will be resolved by discussion. Table 1 Items of data extraction.

Items Content
General

Assessment of methodological quality
To assess the methodological quality of each SR, 2 independent investigators will use the assessment of multiple systematic reviews-2 (AMSTAR-2) instrument for assessment. AMSTAR-2, the updated version of original AMSTAR, can be used to appraise SRs of randomized and nonrandomized studies of health care interventions. [22][23][24][25] It contains 16 items, among which 7 are critical domains, and each item will be judged as "Yes," "No," or "Partial Yes." The overall quality of each SR will be explicitly classified as high, moderate, low, and critically low according to the critical domains. Discrepancies between reviewers will be resolved by discussion or by a third reviewer if consensus cannot be reached.
2.6. Data analysis 2.6.1. Evidence map. We will create a bubble plot to summarize the main characteristics and quality of each SR. The size of the bubble will represent the number of participants, the color of the bubble will represent the methodological quality of SRs, the xaxis will denote the name of biomarkers, and the y-axis will reveal the number of SRs.
2.6.2. Pairwise meta-analysis. The pairwise meta-analysis will be conducted with STATA (13.0; Stata Corporation, College Station, TX) using the data of sensitivity, specificity, DOR, positive likelihood ratio, negative likelihood ratio, and their 95%CI lower limit, 95%CI upper limit extracted from each original study of the SRs. The analysis will be performed using the Mantel-Haenszel statistical method with a random-effects model. To assess the heterogeneity among studies, we will use the chi-squared test and the I 2 statistics. The I 2 statistics of 25%, 50%, and 75% will be considered as cut-off points for low, moderate and high degrees of heterogeneity, respectively. If we find considerable heterogeneity among the studies, we will conduct subgroup analyses to explore the sources of heterogeneity.
2.6.3. Adjusted indirect comparisons. The indirect comparisons will be conducted with the data of relative sensitivity, relative specificity, and relative DOR between different biomarkers calculated by STATA (13.0; Stata Corporation).
2.6.4. Subgroup analysis. We will identify all the primary studies that reported results of subgroup analysis and extract data from these studies. If there is enough data available from the primary research. We will perform subgroup analyses according to the sex, age, and weight of patients; the country of the study; and the cutoff of biomarkers.
2.6.5. Assessment of publication bias. Potential publication bias will be assessed using the Egger test and funnel plot for results with greater than or equal to 10 studies.

Results of study selection
The electronic searches identified 783 potentially relevant records, among which 780 identified through database searching and 3 identified through the manual screening. After reviewing the titles and abstracts, 553 records were excluded and 123 SRs were selected for retrieve full-texts. After full-text evaluation, 88 SRs were entered in the quantitative synthesis. Among the remaining 35 excluded SRs, 5 were not on CRC, 6 were not assessed the diagnostic value of biomarkers, 4 were assessed the prognostic value of biomarkers, 15 were abstracts or reviews, and 5 SRs failed to extract detailed data. The PRISMA flow chart of literature section is presented in Figure 1.

Ethics and dissemination
Ethical approvals and patient consent are not necessary because this is an overview based on published SRs. The findings of this project will provide a general overview and evidence of the diagnostic value of biomarkers in detecting CRC. The results will be submitted to a peer-reviewed journal for publication. We hope that these findings will help clinicians and patients choose a more appropriate method for rectal cancer diagnosis.


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Mycoflora, Proximate Composition and Mineral Analysis during the Storage of Smoked Dried Crayfish (Penaeus natialis - Shrimps)

This study was carried out to assess the changes in proximate composition, mineral content and mycoflora associated with smoked dried crayfish Penaeus natialis (shrimps) stored for twenty weeks. Smoked dried crayfish Penaeus natialis (shrimps) were purchased at Igbokoda, Ilaje Local Government Market, Ondo State, Nigeria. They were studied under storage for twenty weeks (6 months) and the proximate, mineral and mycofloral analyses were carried out at four weeks interval. The mycoflora were isolated using direct plating and dilution methods on Potato Dextrose Agar (PDA), Saboraud Dextrose Agar (SDA) and Malt Extract Agar (MEA) and identified using their cultural and morphological features with reference to standard procedures accordingly. The fungi isolated using direct plating methods and dilution methods were Aspergillus niger, Aspergillus flavus, Aspergillus fumigates, Rhizopus sp., Phytophthora siskiyouensis, Penicillum sp. and Mucor sp. The proximate analysis result showed a decrease in Ash, fat, and crude fibre content while moisture, crude protein and carbohydrate content increased respectively during the twenty four weeks storage. The mineral analysis result of the smoked dried crayfish Penaeus natialis (shrimps) showed a decrease in Sodium, Potassium, Calcium, Magnesium, Zinc, Iron, Copper, Manganese, Cadmium and Phosphorous respectively. This study showed that the smoked dried crayfish Penaeus natialis (shrimps) were contaminated by fungi; which is an indication that the market places where these products were displayed for sale were not hygienic coupled with leaving the products in open air without coverage which could allow products contamination with fungal spores leading to fungal spores germination, deterioration and spoilage of products during storage.  Good hygiene, constant product checking and sensitization of the products processors, handlers and sellers will minimize exposure to fungal spores’ contamination while mitigating deterioration and spoilage of the products during storage.

INTRODUCTION
Crayfish Penaeus natialis (shrimps) is an important flavour ingredient in many Nigerian local preparations. Crayfish are eaten worldwide like other edible crustaceans, only a small portion of the body of a crayfish is eaten in most prepared dishes, such as soups, bisques, only the tail portion is served [1]. Crayfish processing has become a large part of the crawfish industry. Crawfish processing is a modern industry that produces a high quality product available for consumption world-wide [2,3].
Preservation of crayfish Penaeus natialis (shrimps) is very important because it is easily susceptible to deterioration immediately after harvest and to prevent economic losses. The development of machinery that could be employed for effective handling, harvesting, processing and storage of sea foods such as fish and crayfish cannot be over-emphasized especially when aquaculture is growing fast in Nigeria [4]. The use of smoke in local fish preservation was reported by Eyo [5] and the implication of poor postharvest handling of crayfish has also been reported Kumolu-Johnson et al. [6]. Smoke drying is done to partially cook, remove water, obtain brown colour, improve organoleptic flavor and control microbial and enzymatic actions that may cause spoilage. Preservation effects of smoke derived from the antioxidant and antimicrobial properties of its phenolic compound have been reported by Shehu et al. [7] and Abou-zaid and Mohammed [8]. In local markets, crayfish is retailed open as small heaps on tables to attract consumers and information on duration of effectiveness of smoke drying on crayfish quality is scarce. The essence of processing is to preserve and stop microbial deterioration action on food and to retain the quality of the food [9]. However, there is little or no adequate information on the effectiveness of smoke drying on crayfish quality; hence this study is aimed at studying the changes in proximate composition, mineral content and mycoflora associated with smoked dried crayfish Penaeus natialis (shrimps) during twenty four weeks storage.

Collection of Samples
Samples of Crayfish namely Penaeus natialis (shrimps) were randomly purchased at Igbokoda, Ilaje Local Government Market, Ondo State, Nigeria. The dried crayfish Penaeus natialis (shrimps) samples were clearly-labeled, stored at room temperature in a sterile airtight container, and kept in a well-ventilated laboratory for a period of twenty four weeks (6 months) under investigation.

Mycoflora Isolation from the Stored Smoked Dried Crayfish Penaeus natialis (Shrimps)
The mycoflora associated with smoked dried crayfish Penaeus natialis (shrimps) during storage were isolated using the methods described below.

Direct plating method
Visible mouldy sundried crayfish Penaeus natialis (shrimps) were examined and randomly selected from the stored samples for mycofloral isolation using the method described by Amusa [10]. The sample surfaces were sterilized with ethanol and washed in sterile distilled water. The sterilized samples were aseptically placed on Potato Dextrose Agar plates with sterilized spatula and incubated at 28°C for 5days. The hyphae tips of each fungal growth were successively sub-cultured on freshly prepared Potato Dextrose Agar plates until pure colonies were obtained [11]. The cultures were examined microscopically to assess the fungi present.

Dilution plate method
The dilution plate method was done by placing 1 g of smoked dried crayfish Penaeus natialis (shrimps) in sterile distilled water and shaken thoroughly. One ml each of the standardized sample was pipette into 9 ml of sterile distilled water in test tube; and serially diluted in series of test tubes containing sterile distilled water. One ml each of aliquots of 10 -2 and 10 -3 was introduced into molten Potato Dextrose Agar (PDA) plates in duplicates for each isolate and incubated at 28°C for 5 days. The fungal growths were observed every 24hours for the fruiting bodies and hyphae tips of each fungus were sub-cultured successively until pure cultures were obtained [11]. The cultures were examined microscopically to assess the fungi present.

Identification of Mycoflora
The mycoflora isolated from the stored smoked dried crayfish Penaeus natialis (shrimps) were identified by their gross cultural and morphological features. The mycoflora were examined under bright day light for colour of the culture and further examination were carried out using Needle mount preparation method as described by Tuite [12], Crowley et al. [13] and Egbebi et al. [11] and Slide culture technique method as described by Fagbohun et al. [14].

Proximate analysis
Samples of the stored smoked dried crayfish Penaeus natialis (shrimps) were analysed for the for ash, crude fiber, moisture and fat contents according to the methods described by Pearson [15] and A.O.A.C. [16]. The nitrogen was determined by Micro-Kjeldahl method as described by Pearson [15] and the percentage nitrogen was converted to crude protein by multiplying 6.25. The carbohydrate content was estimated by the difference in value obtained when all the chemical composition values were subtracted from 100%. All determinations were in triplicates and values of each constituent were expressed in percentage.

Mineral analysis
The stored smoked dried crayfish Penaeus natialis (shrimps) were analysed for the minerals using the solution obtained by dry ashing the sample at 550°C and dissolving it in 10% HCL (25 ml [16]. The detection limits had precisely been determined using the methods of Varian Techtron [17] as Mn 0.01, Cu 0.005, Co 0.05, Zn 0.005, Fe 0.02, Mg 0.002, Ca 0.04, Na 0.001, ppm (all for aqueous solutions). The optimum analytical range was 0.5 to 10 absorbance units with coefficient of variation of 0.05-0.40%. Phosphovanadomolybdate method using a spectronic 20 colorimeter (Galenkamp, London, UK) (AOAC) [16]. All chemicals were BDH analytical grade.

RESULTS AND DISCUSSION
The proximate content of smoked dried crayfish Penaeus natialis (shrimps) during twenty four weeks storage is shown in Table 1.
The mineral content of smoked dried crayfish Penaeus natialis (shrimps) during twenty four weeks storage is shown in Table 2.
The mycoflora isolated from smoked dried crayfish Penaeus natialis (shrimps) during twenty four weeks storage is shown in Table 3.

Proximate Analysis
There was a significant decrease in ash content (13.45-10.29 g/100 g), fat (5.10-3.89 g/100 g), moisture content (6.40-6.21 g/100 g), crude protein (68.46-68.07 g/100 g) and there was an increase in carbohydrate (6.40-11.53 g/100 g) while crude fibre was not detected during the twenth four weeks storage of smoked dried crayfish Penaeus natialis (shrimps)as shown in Table 1. This result is in agreement with the findings of Girard [18] who reported a significant reduction in ash content of cattle hide from (1.67-0.83) mg/100 g after storage for months. Decrease in ash content indicates loss of nutrients as the storage progressed. Ash content in food contributes to the residue remaining after all the moisture has been removed as well as the organic material (fat, protein, carbohydrates, vitamins, organic acid etc.) have been incinerated at a temperature of about 500°C. Ash content is generally taken to be a measure of the mineral content of the original food [19]. However, this result contradicts that of Oladejo and Adebayo-Tayo [20] who reported an increase in crude protein (21.68-54.16) mg/100 g of "Banda" dried meat during storage and Rodolfo et al. [21] who found out that fungi increase the protein content of the samples on which they grow. This result is also different from the findings of Lawal et al. [22] who reported a decrease in the proximate content such as carbohydrate content of sundried coco yam chips during storage. Crude fiber was not detected in stored smoked dried crayfish (shrimps) which is similar to that of Eleazu [23] who reported that crude fiber was not found in the 10%, 30%, or 40% NRCRI cassava bread samples or in the 100% wheat bread. There was a reduction in the moisture content from 6.40 -6.21 (g/100 g). This result is in agreement with the work of Ajai et al. [24] who reported a decrease in the moisture contents of milk samples after storage from (8.32-7.51) g/100g. It is known that products that have low fat values normally have high moisture contents. Decrease in water content in this study could be attributed to the fact that infecting fungus utilizes the moisture content for its survival and growth. The shelf life of any product is influenced by the amount of water present in it [25]. Moisture content is a widely used parameter in the processing and testing of food. It is an index of water activity of many foods and determines the shelf life or keeping quality of the food. The observed value in this study implies that smoked dried crayfish (shrimp) will have a long shelf life because of the low moisture content.

Mineral Analysis
The summary of the mineral composition of smoked dried crayfish penaeus natialis (shrimps) during twenty four weeks storage showed a decrease in Sodium (58.90-56.56 mg/100 g), Potassium (66.30-60.39 mg/100 g), Calcium (86.65-81.29 mg/100 g), Magnesium (49.90-45.80 mg/100 g), Zinc (0.63-0.48 mg/100 g), Iron (6.33-5.55 mg/100 g), Copper (0.28-0.09 mg/100 g), Manganese (0.99-0.54 mg/100 g), cadmium (0.23-0.06 mg/100 g) and Phosphorous (106.10-100.89 mg/100 g) as shown in Table 2. This result supports the findings of Oladejo and Adebayo-Tayo [20] who reported a reduction in Sodium (0.35-1.55) mg/100 g in "Banda" dried meat during storage. This result is in contrast to the work of Hassan et al. (2005), who reported an increase in sodium content of Vernonia amygdalina leaf protein concentrates of (57.5±0.34 mg/100 g). High sodium content in food is of great concern for health because of its implication in high blood pressure [26]. The result of this study indicated that eating of smoke dried crayfish (shrimp) could not lead to high in blood pressure. Low sodium content is beneficial in the treatment of hypertension and renal diseases [27]. The manganese content of stored smoked    The implication of mycofloral in these products could be attributed to the ever increasing demand for smoked dried crayfish Penaeus monodon (shrimps) and in the quest of the retailers to meet this need the fish are overloaded on the smoking kiln during processing; as a result they are exposed to a reduced intensity of heat for short period of time. This leads to improper processing and vulnerability of the fish to fungal contamination [30]. The market place where the smoked dried crayfish products are displayed for sale most times are not clean or hygienic, such as in open trays without coverage Hassan et al. [26] Fungi found in stored food are divided into two groups namely the field fungi and the storage fungi. Most at times it is difficult to distinguish between the two as fungal growth may start both in the field and during storage. Species of Aspergillus, Rhizopus and Penicillum have been reported as storage fungi which infect crops on the field and may persist and proliferate in storage [31].

CONCLUSION
This current study indicated that the stored smoke dried crayfish (shrimps) were contaminated with fungal species with significant loss of nutrients during the twenty four weeks storage. Therefore, special attention should be paid to the microbial investigation to minimize the threats posed to public health. The crayfish (shrimps) must be properly dried to reduce the moisture content before packaging to prevent fungal invasion and enhance the good keeping and storage quality. Good sanitary practices including good storage practices must be followed and micro biological standards must be adhered to by checking production procedures and handling until the stored smoke dried crayfish (shrimps) reach the consumer's table. Stored smoke dried crayfish (shrimps) sellers should be sensitized on the importance of good hygienic practices, good housekeeping and proper storage conditions to prevent deterioration of their product.