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In recent years, active learning with group work activities has been adopted in class of educational institution. Group work activities are expected to form students’ independence, cooperativeness, thinking faculty, expressiveness, and so on. However, in group work activities, there are drawbacks that adversely affect the progress and outcomes of students’ activities if an imbalance occurs in the personality and characteristics among the group members. For that reason, in order to maximize the learning effects of group work activities, it is indispensable to balance the performance exerted by the organized group. Therefore, “Formation of Abilities Averaging (FAA)” was proposed as a model of the method of organization to complement a group in consideration of personal characteristics and abilities in this research. Moreover, in order to demonstrate the characteristics of FAA, several methods were compared. In the experiments, the group work activities of Think-Pair-Share was done with a history class of general studies as a model. Then, the superiority was evaluated by the results of questionnaire surveys using Visual Analog Scale after the activities. As a result, in the FAA designating the positions within the group, the interest that leads to self-study after the class was high, and the effect of students’ motivation to learn was observed. In addition, FAA enables the organizer to control the condition of organization, it was possible to make more groups than when using other methods, indicated high degree of freedom and versatility.

This chapter describes a multi degrees of freedom (hereafter, "DOF") motion sensor system in 3D space. There are two major areas where multiple-DOF motion sensors are needed. The first involves vehicles, helicopters or humanoid robots, which are multi-input multi-output objects, each having 3 DOF in all translational and rotational directions. Thus, an accurate measurement of all 6 DOF motions is needed for their analysis and control. The second area involves considering not only the translational components, but also the rotational components for 1-DOF linear motion on ground since this type of motion is always affected to a certain extent by motion along other axes, and mixed signals of translational and rotational components are detected in the case of using 1-DOF motion sensors. So far, inertial navigation systems (INS) with highly accurate gyro sensors and accelerometers have been used in the field of rocketry and aerodynamics. However, it is difficult to install such systems combined with gyroscopes and accelerometers on small robots due to their large size, weight and cost. In recent years, small vibration gyro systems and micro-machine gyro systems have been developed by using MEMS (micro-electromechanical system) technology. However, the sensor units which utilize these systems are not small, weighing about 6 to 10 kilograms, and in addition there are some problems with measurement accuracy and stability. In the case of multiaxial sensors, it is known that the specific problem of non-linear cross effect arises, which means that other axial components interfere with the target one, and the measured values differ from the true values due to this effect. As a result, the cross effect reduces the measurement accuracy. In addition, as a result of increasing the effect by the gain and offset errors of each axis, stability is lost. Therefore, sensor calibration is required for suppressing the cross effect, as well as for decreasing the gain and offset errors. However, in general, gyro systems cannot be calibrated in isolation, which needs special equipments, such as an accurate rotary table, for generating a nominal motion. Thus, a proper calibration of the multiaxial sensors by the user is extremely difficult. In the present work, we propose a newly developed 6-DOF motion sensor using only multiple accelerometers, without the gyro system. The advantage of using accelerometers is that they can be calibrated with relative ease, using only the gravitational acceleration without any special equipment. So far, we have performed several experiments using the

Safflower is a crop cultivated in the Middle East and is an annual plant of the Asteraceae family with a height of 0.5 to 1.0 m and a radius of 2.5 to 4.0 cm in early summer. Safflower is used for high-quality local foods cosmetics, dyes, but the harvesting is done by hand, therefore the work efficiency is low. In this study, we developed a cultivation management system to increase the efficiency of the process; from cultivation to harvest, aimed at revitalizing the production of safflower. Advancements in Artificial Intelligence and robotics has allowed for the exploration remote sensing methods that gather and analyses large amounts of data to guide precision management. A detecting unit for buds and flowers was developed using AI(Deep Learning) to count and analyze the location. This data is stored inGIS applicaiton to manage the growth of buds and flowers. The flower location data is used by the AI harvesting robot and robot arm controller to guide the harvesting unit to the flower. The harvesting unit cut the petals by rotary cutter, then vacuum the petals into a storage tank. Further work is aimed at improving the harvesting performance and going to enable the new feature of autonomous driving and harvesting in field. And it can be used as AI harvester has multi-feature robot as ECOSYTEM for variety of plants and crops, fruits precision management. We hope to help the elderly farmers to keep the production of safflower in Yamagata, Japan.

Recent almost developed countries like Japan, senior people are increasing. In such as aging society, supplying human resource to care the senior people doesn't catch up with its demands. Therefore, the societies encourage the cared elderly people become to manage to live by themselves in their life. To realize such as the society of independent elderly people, many assistive machines and tools are developed. In particular, electric wheelchairs are very effective to keep and improve their Quality of Life (QOL). In this study, to promote the use of electric wheelchairs, a novel operation system for electric wheelchair using pointing device (Hands-Free Operation-Interface) is proposed. The proposed system can operate an electric wheelchair without body action. To investigate operation system of the wheelchair, its driving performance is compared with driving using a standard joystick under same slalom course running. There were some obstacles in the slalom course, driver needed to drive the electric wheelchair and make own way around these obstacles to goal. In this experiment, success-ratio and running time are compared between Hands-Free Operation-Interface and standard joystick operation to evaluate the operability of the systems. As a result, it is suggested that success-ratio is depended on operators, and the average of success-ratio is 67 %. Also running time is observed that depended on driving ability of operators. Therefore, these results showed that practice and adapting of users are necessary to drive the electric wheelchair using the proposed operation system. In addition, this study showed some direction for controlling the system safely.

In recent years, active learning with group work has been adopted in educational institution lessons. Application of group work is expected to create student’s independence and cooperativeness, thinking faculty, expressiveness and so on. However, in group work, there are drawbacks that adversely affect the progress and outcomes of activities if an imbalance occurs in the personality and characteristics among members of the group. Therefore, in order to maximize the learning effect of group work, balancing the performance exerted by the organized group is indispensable. In this research, a complementary group organization model “Formation of Ability Averaging” (FAA) considering characteristics and problems of individuals was proposed. Also, in order to demonstrate the characteristics of capacity averaging organization, several organization methods were compared. In the experiment, Think-Pair-Share group work was executed with the history of general studies as a theme. The superiority was evaluated by questionnaire survey using Visual Analog Scale after activity. As a result, in the Ability Averaging Formation designating the positions within the group, the interest that leads to self-study after class was high, and the effect of student’s motivation to learn was observed. In addition, FFA was able to determine the condition of organization by the organizer, so the number of groups organized was larger than that of the other, indicating high degree of freedom and versatility.

Engineering Ethics is an example of a subject in which there is no single correct answer. Unlike most other courses in the engineering field, where problems have a unique answer, students need to derive an optimal solution according to the context. For the ethics cource, the objective is being able to apply the ideas in practice in the real world, and this should be more of a focus than being able to understand the specialist terminology and derive model solutions. Group discussions and analysis of past cases are effective ways of cultivating this kind of ethical sense. In this study, we used an audience response system in classes and evaluated the learning benefits. Having students' own responses visualized in real time encourages active learning while still maintaining continuity of instruction. In particular, in the group presentations for ethics case studies, peer evaluations of the presentation techniques as well as the content of the idead presented were used. Having the voting results visualized in real time was useful for encouraging students to take an active interest in listening to the presentations. This was an effective way to encourage students to come to their own conclusions wnile understanding the diverse range of values held by other people.

This paper describes a new multi-degrees-of-freedom (DOF) motion sensor system in three-dimensional space. Our sensor system detects the acceleration of moving objects. In general, the moving objects each have 3 DOFs in the translational and rotational directions. Thus, the accurate measurement of all 6 DOFs of motion is essential to control these objects. We have developed a new acceleration sensor that measures 6-DOF motions by resolving multiple acceleration signals and are currently investigating its performance. In the case of multi-axial sensors, a specific problem of cross effect arises, which means the value obtained is different from the actual value, so sensor calibration becomes important. In this work, we achieved relatively stable measurement by inhibiting the cross effect through calibration. Though the only physical value that our sensor system can measure directly is 6-DOF acceleration, we investigated the angular velocity to compare with a conventional highly accurate gyro sensor. In calculations of angular velocity, we combined the motion characteristic with a Kalman filter and estimated angular velocities from detected accelerations. Our method generates a required observational signal in the Kalman filter artificially, using detected translational accelerations with gravity components and non-periodically refers to the observational signal. Finally, we investigated the proposed method in an experiment involving vehicle motion especially likely to have cross effect, and demonstrate that our sensor system (6-DOF accelerometer) performs well.

This paper investigates an efficient calibration method of 6 DOF acceleration sensor systems using multiple dual-axis accelerometers. So far, we have developed a calibration method to our system as sensitivities of 6 accelerometers were independent each other. But, actually, in dual-axis type accelerometers, there exist some dependency among each axis-sensitivities. Then, we reconstruct a new calibration method to above case, and investigate the influence of the error marging included in calibration. As a result, it is shown that the geometrical errors to be identified which absolute value is small or near 0 compared with other should be assumed 0 from the beginning, and we can decrease the number of calibration data and improve the efficiency of the calibration.

There is a method of measuring a shake and vibration quantiatively and evaluating, in one of the performance evaluations of a moving object like vehicles. In that case, the acceleration information as an amount of physics of a shake and vibration becomes important. Furthermore, the acceleration information on 6 DOF is needed when measuring complicated motion of vehicles etc. Then, we constructed 6 DOF acceleration sensor system which used three dual-axis accelerometers, and enabled measurement 6 DOF acceleration. In this paper, we analyzed the problem of the stability in the measurement system of the same method that was not clearly analyzed in the past research. We took notics of a nonlinear term that consist of an angular velocity, which included in the derivation algorithm of 6 DOF acceleration, and made clearly how a nonlinear term would influence the stability of a measurement system by the analysis simulation. And we installed the acceleration sensor in vehicles and performed measuring 6 DOF acceleration to a slalom run. As a result of comparing survey data with a simulation data, emission of the measurement system under the influence of a nonlinear term was not seen.

Tryptophan (Trp) is an essential amino acid (AA) and it may be limiting to ruminants when feed rations are based on corn (maize). Distiller's grains (DGs) from barley are the dried residue remaining after the starch fraction of corn is fermented using selected yeasts and enzymes to produce alcohol. DG is a rich source of nutrients, because it contains polyphenol, peptides, yeast extracts (nucleic acids and amino acids), citric acid, fermented barley extract and fiber. The ionophore-type feed additives (monensin and salinomycin) modify the movement of monovalent (sodium and potassium) and divalent (calcium) ions across biologic membranes, modify the rumen microflora pattern, decrease acetate and methane production, increase propionate, may improve nitrogen utilization and can increase dry matter digestibility in ruminants. Vitamin B6 (B6) acts as a coenzyme in many enzymatic reactions that are involved in the metabolism of AA. An in vitro study was conducted to examine the effects of salinomycin (SL), monensin (MN), B6 and DG on the production of tryptophan (Trp) and other related compounds from indolepyruvic acid (IPA) by mixed ruminal bacteria (B), protozoa (P) and their mixture (BP). Ruminal microbial suspensions were anaerobically incubated at 39 °C for 12 h, and analyses of Trp and its related metabolites were carried out by HPLC. When 1 mM of IPA was used as a substrate, it was mainly converted to Trp and indoleacetic acid (IAA), resulting in concentrations of 300 and 131, 410 and 28, and 435 and 112 μM in B, P and BP suspensions, respectively. A considerable amount of skatole (SKT) (in B and BP) and indolelactic acid (ILA) (in B, P and BP), and small amount of p-cresol (CRL) (in B and BP) were also produced from IPA. The production of Trp from IPA with SL, MN, B6 and DG were increased (P < 0.05) by 18.9–55.3, 9.0–50.4 and 5.1–16.1% in B, P and BP, respectively. The increased Trp production with these additives occurred by the simultaneous inhibition of IAA from IPA. On the other hand, addition of B6 into B, P and BP enhanced (P < 0.05) IAA from IPA by 18.9, 26.5 and 5.1%, respectively. Trp produced from IPA with SL was 13.8, 8.8 and 5.0% higher than that with MN in B, P and BP, respectively. Salinomycin and MN completely inhibited the SKT and CRL (in B and BP), and partially inhibited ILA (in B, P and BP) from IPA. On the other hand, B6 stimulated the production of SKT and CRL in B and BP, and ILA in B, P and BP from IPA. No effects were observed on the production of SKT, CRL and ILA by DG. We conclude that addition of these additives to feeds may increase duodenal Trp supply in ruminants. In addition, SL and MN may also inhibit the production of clinically important Trp metabolites (SKT, CRL and ILA).

An anaerobic in vitro study of tyrosine (Tyr) metabolism by rumen bacteria (B), protozoa (P) and their mixture (BP) was conducted using an HPLC analytical method to obtain some basic and systematic information on aromatic amino acid metabolism in the rumen. Most of the Tyr (> 96%) disappeared after 12 h incubation in B and BP, but only 58% in P. Tyr was converted mainly to p-hydroxyphenylacetic acid (HPA) in all microbial suspensions. About 45% of disappeared Tyr in B and P, and about 35% in BP were converted to HPA. An appreciable amount of phenylalanine (Phe), 13 and 3% of disappeared Tyr, and a small amount of tryptophan (Trp), 8 and 1% of disappeared Tyr, were also produced from Tyr by rumen bacteria and protozoa, respectively. Small amounts of p-hydroxyphenylpyruvic acid (about 4 and 6% of disappeared Tyr) were produced from Tyr in B and P, respectively. A moderately large amount of phenylacetic acid (14% of disappeared Tyr) was produced from Tyr in P which was 1.9 times higher than that in B. Phenylpropionic acid and p-hydroxybenzoic acid were produced only in B and BP. It was concluded that the Tyr degradation ability of rumen bacteria was about 1.5 times higher than that of rumen protozoa. Degraded Tyr mainly produces HPA and then two other aromatic amino acids, Phe and Trp, which are considered essential amino acids for ruminants. Therefore, it is speculated that the requirement for Phe and Trp in ruminants may be partially fulfilled if Tyr is sufficiently supplied in rations.

In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice-cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl2 to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.

Three segments of cattle small intestine (duodenum, upper jejuno-ileum and lower jejuno-ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno-ileum and lower jejuno-ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at −20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen-produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.

It has been demonstrated that L-pipecolic acid (L-PA), a major metabolic intermediate of L-lysine (L-Lys) in the brain, is involved in the functioning of the γ-aminobutyric acid-ergic system. Previous study has shown that intracerebroventricular injection of L-PA suppressed feeding and induced sleep-like behavior in neonatal chicks. The present study examines whether the action of L-PA was induced by gavage in both chicks and mice. Oral administration of L-PA significantly inhibited food intake at 2 h after treatment in neonatal chicks, although no significant effect of L-Lys was detected. In mice, oral L-PA suppressed food intake compared to the control after 2 h of treatment. It was concluded that L-PA was effective for suppression of food intake after oral administration in both avian and mammalian species.

An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO(2)). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h with or without MSO (1 mM) or MSO(2) (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 micromol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO(2) and 2-aminobutyric acid (2AB) in B and BP. MSO(2) as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO(2); but MSO can be utilized by B and BP for producing Met.

The catabolism of arginine (Arg) by mixed rumen bacteria (B), mixed rumen protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system in order to confirm the metabolic pathway of Arg and provide basic information for enzymatic and molecular studies as well as an understanding of the quantitative distribution of metabolites. Rumen microbial suspensions (B, P, and BP) collected from fistulated goats were anaerobically incubated with or without 1 mmol/L Arg at 39°C for 12 h. Arg and other related compounds such as citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV) in both supernatant and hydrolyzates of B, P, and BP suspensions were analyzed by HPLC. The metabolic pathways of Arg in mixed rumen bacteria and mixed rumen protozoa were considered to be as follows: rumen bacteria, Arg Cit Orn Pro 5AV VFAs + NH3; rumen protozoa, Arg Cit Orn Pro 5AV. The disappearance of Arg (1 mmol/L) was approximately 52.9 and 88.2% in B, 33.9 and 55.6% in P, and 52.8 and 85.2% in BP during 6 and 12 h incubations, respectively. When expressed in units of ‘per gram (g) of microbial nitrogen (MN)’, the net degradation rate of Arg in BP (50.3 µmol/g MN/h) was approximately 46% lower than that of B during a 12 h incubation period. The presence of protozoa tended to inhibit the production of Orn from Cit and the production of 5AV from Pro which were thought to be rate-limiting steps of Arg metabolism in rumen microorganisms. As a result, protozoa appeared to have a saving effect on Arg metabolism, that is, protozoa protected Arg from wasteful exhaustion in the rumen.

Acetone, which is produced from butyric acid when it passes through the rumen wall, was infused into the rumen and jugular veins of three female goats to investigate the role of acetone in ruminating and masticating behavior. The ruminating behavior, as measured by the number of boli and the ruminating time, decreased (p

Concept and establishment of essential amino acids in animals and human beings rendered immeasurable contributions to animal production and human health. In ruminant animals, however, essential amino acids have never been completely established. The present review proposes a hypothesis that histidine may not be an essential amino acid for normal growing cattle (Japanese black) at least at the growing stage after about 450 kg of body weight on the basis of the experimental results of histidinol dehydrogenase activities in some tissues of the cattle together with hints from which the hypothesis was derived. At the same time, histidinol dehydrogenase activities in liver, kidney and muscle of swine, mouse, fowl and wild duck will be shown and the essentiality of histidine in these animals will be discussed. Finally, the essentiality of histidine for adult human will briefly be discussed. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 3 : 445-454)

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species-enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L-lysine (1.0 mmol/L L-Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L-Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L-Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D-Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D-Lys, though at a lower rate (1/3) compared with L-Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L- or D-Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D-Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen-produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.

Biosynthesis of arginine (Arg) from citrulline (Cit), ornithine (Orn), proline (Pro), and 5-aminovaleric acid (5AV) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system from the standpoint of protein nutrition in ruminants. Rumen microorganisms, collected from ruminally fistulated goats, were anaerobically incubated with or without 1 mmol/L each of substrates at 39°C for 12 h. Arginine and other related compounds, produced in both supernatants and acid-hydrolyzates of microorganisms in B, P, and BP suspensions, were analyzed by high-performance liquid chromatography. Arginine production from Cit in BP, when expressed with a unit of ‘μmol/g microbial nitrogen’, was approximately 70% and 94% higher than that in B and P, respectively, in a 12-h incubation period. In the case of Orn, the values were approximately 30% and 75%. Both rumen bacteria and protozoa could produce Cit and Orn from Pro, so it is assumed that they can produce Arg from Pro. Rumen protozoa were unable to degrade 5AV and it was the final product in the metabolism of Cit, Orn and Pro in P suspension. A trace amount of Orn and Pro produced from 5AV in B and BP suspensions indicated that the reversible reaction of 5AV formation was performed only by rumen bacteria. This is the first quantitative report on Arg biosynthesis from its precursors by rumen microorganisms.

The purpose of this study was to determine the optimal ratio of amino acids, in terms of the ratio of non-protein calories to nitrogen (NPC/N ratio), in peripheral parenteral nutrition (PPN). Test solutions that NPC/N ratio is 50, 100, 150 or 200, or amino acid free solution were administered parenterally at a rate of 120 kcal/kg/day for 5 days to normal rats, after which the effects of amino acid on protein metabolism were assessed. Cumulative nitrogen balance increased with a decrease in the NPC/N ratio (with a increase in amino acid dose), but there were no statistically significant differences between group NPC/N: 50, group NPC/N: 100, and group NPC/N: 150. The protein synthesis rate in the liver increased with a decrease in the NPC/N ratio, and the value in group NPC/N: 200 and group NPC/N: 150 was significantly lower than that in group NPC/N: 50. However, in group NPC/N: 50, the levels of serum urea nitrogen (SUN) and serum branched chain amino acids (BCAA) were high, suggesting the excessive administration of amino acids. Based on the above findings, the optimal NPC/N ratio is estimated to be about 100 (50 g as amino acids), when the intravenous solution is administered at the anticipated daily dose in clinical use (1000 kcal/day).

Prevotella species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify and investigate properties of one of the enzymes involved in amino acid metabolism by Prevotella bryantii B 1 4, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 51-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with Phenyl-superose, DEAE-Toyopearl 650 M, Sephacryl S-100 HR and Sephadex G-100. The molecular mass of GPA was estimated to be 28.0 kDa by SDS-PAGE. The optimum pH was 6.5 and the activity declined above pH 9.0. GPA was reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, while SDS, EDTA and some heavy metal ions also inhibited activity. The purification and characterization of enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by P. bryantii B 1 4.

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne ( Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39 degrees C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3-12% of the disappeared HPY) and Trp (2-10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2-6% of the disappeared HPY) were produced only in B.

A selective and simple HPLC procedure has been developed to determine histidine (His) and histidinol (HDL) in liver supernate. The separation was performed on a column, Mightysil RP-18 GP. The eluted analytes were measured with UV detection without derivatization which provided detection limits of 1.1 and 2.0 microM for His and HDL (S/N ratio, 3:1), respectively. Recovery of the analytes added to liver sample was 98.3-101.6% within a 1-day study and 95.7-98.6% on different day (6 days) studies. The apparent histidinol dehydrogenase activities (nmol/g wet tissue) at pH 8, 9, 10, 11, and 12 were 38.6, 50.4, 160.3, 274.3, and 185.6 for liver; 90.6, 132.2, 30.7, 22.1, and 6.76 for kidney; 0.0, 0.0, 38.2, 20.1, and 12.9 for pancreas; 0.0, 0.0, 0.0, 14.7, and 6.8 for spleen; 0.0, 0.0, 4.2, 6.8, and 0.0 for muscle; and 0.0, 0.0, 4.9, 1.8, and 0.0 for small intestine, respectively. On the basis of optimum pH values, histidinol dehydrogenase activity in the organs was in the following order: liver>kidney>pancreas>spleen>muscle>small intestine.

Isopropyl alcohol (IPA), produced from acetone by rumen bacterial action, was infused into the rumen of three female goats kept in a climatically controlled experimental room during feeding to investigate the mechanism and roles of IPA in ruminating behavior (number of boli and ruminating time). The ruminating behavior measured by the number of boli, ruminating time, number of remastications, and remasticating time increased (p

This study quantitatively investigated the biosynthesis of methionine (Met) and the production of related compounds from homocysteine (Hcys), cystathionine (Cysta), and homoserine (Hser) plus cysteine (Cys) by rumen bacteria (B) or protozoa (P) alone and by a mixture of these bacteria and protozoa (BP). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h. Hcys, Cysta, and Hser plus Cys were catabolized by all rumen microbial fractions to different extents. B, P, and BP converted Hcys to Met with 2-aminobutyric acid (2AB) and methionine sulfoxide. The Met-producing ability of B (83.2 micromol g(-1) microbial nitrogen; MN) from Hcys was about 3.6 times higher than that of P in a 6-h incubation period. The ability of BP, during the same incubation period, was about 30.0% higher than that of B. Hcys, Met, and 2AB were formed when Cysta was incubated with B, P, or BP. Rumen microbial fermentation of Hser plus Cys led to the formation of Cysta, Met (through Hcys), and 2AB. Thus the results indicated that a trans-sulfurylation pathway for Met synthesis was operating in the rumen bacteria and protozoa. The results mentioned above have been demonstrated for the first time in B, P, and BP in the present study.

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39 degrees C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 micromol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 micromol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report.

The authors evaluated the cell growth inhibition, reduction of tumourigenicity, and differentiation-inducing effects of sodium phenylacetate (NaPA) on a canine mammary tumour cell line. Treatment of the canine mammary tumour cell line (MCM-B2) with NaPA lead to the arrest of cell growth. Sodium phenylacetate induced changes in the cells to non-malignant characteristics, as indicated by a reduction of colony formation in semi-solid agar and a decrease in tumour formation in athymic mice. Moreover, NaPA induced morphological changes from a spindle-shaped to an epithelial-like appearance, and significant accumulation of lipid droplets in the cytoplasm. Immunohistochemically, these treated cells reacted clearly with the antibody for keratin/cytokeratin. Sodium phenylacetate treatment increased the expression of the milk-specific genes α-lactalbumin and β-casein. The results of this study warrant an evaluation of NaPA in a clinical trial to establish its possible value as adjunctive treatment of malignant canine mammary tumours.

The present experiments were conducted to examine if histidinol dehydrogenase (HLDase) activity could be detected in the liver and kidney of cattle, in order to assess the possibility that cattle synthesize histidinc (His) de novo. Crude enzyme solutions of liver and kidney from Japanese black cattle showed HLDase activities with pH optima of 11.0 and 9.0, respectively. This is the first report of HLDase activity in animal tissues. The amount of His produced from histidinol (HDL) by the enzymes of the liver and kidney were calculated to be sufficient for meeting the His requirement of the cattle, based on the crude protein requirement for the animals and His content in muscle protein. In contrast, HLDase activities in swine liver and kidney proved to be insufficient to form sufficient His from HDL to meet their requirement as indicated in the Japanese Feeding Standard for Swine (1998).

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39 degrees C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 micromol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated.

The purpose of this study was to determine the optimal fat proportion to glucose in peripheral parenteral nutrition (PPN). PPN solutions consisting of dextrose + amino acids + fat (0%, 10%, 20%, 30%, 40% or 50% of total energy as fat) were administered parenterally at a rate of 120 kcal/kg/day for 5 days to normal rats, after which the effects on the nutritional status were assessed. Body weight change and cumulative nitrogen balance during the 5 day PPN period tended to increase with increasing fat dose until the 40% fat group. Serum triglyceride (TG), phospholipid (PL), free fatty acid (FFA) and total cholesterol (T-CHO) levels increased with increasing fat dose. Specifically, PL, T-CHO and FFA levels in the 50% fat group were especially higher than that in the normal group. Although the rats were administered with the same composition and dose of amino acid, serum Thr, Trp, Ala and Pro levels tended to decrease with increasing fat dose, with serum Ala and Pro levels in the 50% fat group being lower than that in the normal group. Hepatic glycogen content also tended to decrease with increasing fat dose. These results suggest that gluconeogenesis in the liver is increased in the 50% fat group due to low dextrose dose. In the 40% fat group, although serum PL and T-CHO levels were significantly higher, hepatic glycogen content and serum Ala levels were not significantly different compared with 0% fat group. It was therefore concluded that the optimal fat proportion in the rats receiving an energy intake below the basal energy requirements is 40% of total energy as fat.

A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO2), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150x4.6 mm I.D., 5 microm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (microM) (S/N ratio, 3:1) and quantification (microM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO2, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93-102.3% in the within-day study and 94.52-98.69% on different day (6 days) studies. The average contents (microM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency.

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45–90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (μM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4–103.0% within a 1-day study and 95.4–99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 μM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 μM, respectively, before the morning feeding.

A simple, rapid, and sensitive method was developed for detection and quantitation of lysine (Lys) in various biological samples by isocratic liquid chromatography (LC). Samples containing Lys and other amino acids were derivatized with 9-fluorenylmethyl chloroformate (FMOC-CI). The mobile phase used for isocratic elution was 50 mmol/L sodium acetate buffer (pH 4.20)-acetonitrile (43 + 57, v/v). Lys was detected with a UV detector at 265 nm. The derivatized Lys eluted from a LiChrospher 100 RP-18 (150× 4.0 mm id) column at a retention time of 5.6 min. The limit of detection was 0.73 μmol/L (signal-to-noise [S/N] ratio, 3:1), and the limit of quantitation was 2.37 μmol/L (S/N ratio, 10:1). Lys recoveries from fortified biological samples were >97.5%. Average Lys contents found in rumen fluid samples collected before the morning feeding and at 2.0,4.0, and 6.0 h after feeding were 4.26,3.34,3.58, and 3.82 μmol/L, respectively. The hydrolysate of a sample of mixed rumen microorganisms collected before the morning feeding was determined to contain 1.372 μmol/mg microbial nitrogen in the form of Lys. The Lys concentrations of human plasma, goat plasma, human urine, and goat urine were 140.0, 102.0,58.0, and 32.0 μmol/L, respectively.

Tryptophan (Trp) biosynthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated by using 1 mM of indole-3-pyruvic acid (IPA) as substrate. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds in both the supernatants and the microbial hydrolyzates of the incubation were analyzed by HPLC. As a whole, about 334, 440, and 436mgr;M of Trp were produced from IPA in 12 h by B, P, and BP suspensions, respectively. In the B suspension, a greater portion of synthesized Trp (242mgr;M) from IPA was accumulated as free form in the medium, whereas a large amount of Trp (92mgr;M) was incorporated into cell protein in a 12-h incubation. On the other hand, in the P suspension, a large amount of Trp (475mgr;M) from IPA was also found as free form in the supernatant in a 12-h incubation. Protozoa did not incorporate Trp into cell protein, but they liberated endogenous Trp (34mgr;M) into the medium. The net productions of Trp from IPA were 344.3 and 447.7mgr;mol/g of microbial nitrogen in 12 h by B and P suspensions, respectively. The ability of P to synthesize Trp from IPA was about 30% higher than that of B in 12 h. Trp produced from IPA by B, P, and BP suspensions were simultaneously degraded into its related compounds, and among them, indoleacetic acid (IAA) was a major product found in all microbial suspensions. Productions of IAA were 124, 25, and 99mgr;M from IPA in 12 h by B, P, and BP suspensions, respectively. The formation of indolelactic acid (ILA) from IPA was observed for the first time in all microbial suspensions, and it was about 84, 24, and 54mgr;M in 12 h by B, P, and BP, respectively. Higher IAA and ILA productions were observed in B when compared with P. A small amount of skatole (SKT) (26mgr;M) was produced from IPA in B, whereas a sizable amount of SKT (38mgr;M) was found in BP after a 12-h incubation. p-Cresol (CRL) was also produced from IPA by both B (43mgr;M) and BP (65mgr;M) suspensions in 12 h, and this is also the first discovery to show the formation of CRL from IPA by B and BP suspensions. BP suspension was more active to produce both SKT and CRL from IPA, though P suspension has no ability to produce either SKT or CRL from IPA during a 12-h incubation.

本研究では,トリプトファン(Trp)ならびにその関連化合物としてインドール乳酸(ILA),トリプトフォール(TPP),トリプタミン(TPM),インドール-3-アセトアルデヒド(ICA),インドール酢酸(IAA),インドール-3-アルデヒド(ILD),スカトール(SKT)およびインドール(IND)のほかこれら化合物のピーク出現時間の範囲内に入るp-ヒドロキシフェニル酢酸(HPA)およびtrans-ケイ皮酸(CNM)の高速液体クロマトグラフィー(HPLC)による新迅速定量法を確立した.カラムとしてはLiChrospher 100 RP-18 (250×4mm I.D.)を使用した.また,イソクラティック溶離液の移動相としては,メタノール:50mM酢酸ナトリウム緩衝液(pH4.2)(30:70,v/v)を使用し,ピークはUV検出器(280nm)によりモニターした.分析に先立ち,標準溶液または試料溶液に等量のスルフォサリチル酸(SSA)(4%,w/v)を加えて除タンパク処理を行った後,除タンパク液5μlをHPLCに注入した.最終ピークのSKTは60分頃に出現し,60分以内に上記11種類の化合物の分析が終了した.化合物の回収率は,95%以上であり,また,これらの化合物の検出限界は次のとおり(pmol/5μl): Trp, ILA, IAA,ILDおよびTPP, 19; ICA, HPA, INDおよびCNM, 39; SKT, 156; TPM, 625.次に本法をヤギのルーメン液の分析に応用した結果,HPA, ICA, Trp, ILA, IAA, ILDおよびTPPが検出された.中でも,ICA, HPAおよびIAAは比較的多かった.これらの化合物の濃度は,給餌後の時間により変化した.

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507mgr;M and converted mainly to Phe on the average by 231, 244 and 248mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842mgr;M and converted mainly to PAA on the average by 254, 205 and 461mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (pyridoxine hydrochloride) (B(6)) on the production of lysine from the three stereoisomers of 2,6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12 h with and without DAP-SI (5 mM) as a substrate and SL (5 mg/ml) and/or B(6) (10 mg/ml) as additives. In P suspensions, SL and B(6) reduced the amount of DAP-SI by 2.1 times (p