This article was downloaded by: [Nanyang Technological University] On: 28 April 2015, At: 02:47 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks a
b
a
c
a
d
c
X. Zhang , Z. Sun , F. Cao , H. Ahmad , X. Yang , L. Zhao & T. Wang a
College of Forestry, Nanjing Forestry University, Nanjing, 210037, P.R. China,
b
Department of Animal Husbandry and Veterinary Science, Jiangsu Polytechnic College of Agriculture and Forestry c
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, P.R. China d
College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, Jiangsu Accepted author version posted online: 13 Apr 2015.
Click for updates To cite this article: X. Zhang, Z. Sun, F. Cao, H. Ahmad, X. Yang, L. Zhao & T. Wang (2015): Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks, British Poultry Science, DOI: 10.1080/00071668.2015.1030590 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1030590
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2015.1030590
CBPS-2014-359 MacLeod, March 2015
ip t
Ed. Kjaer, February 2015;
cr
us
capacity, intestinal morphology and microbial ecology in broiler chicks
X. ZHANG, Z. SUN1, F. CAO, H. AHMAD2, X. YANG, L. ZHAO3 AND T. WANG2
1
M
an
College of Forestry, Nanjing Forestry University, Nanjing, 210037, P.R. China,
Department of Animal Husbandry and Veterinary Science, Jiangsu Polytechnic College of
ed
Agriculture and Forestry, 2College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, P.R. China and 3College of Chemical Engineering, Nanjing
ce pt
Forestry University, Nanjing, 210037, Jiangsu
Running title: GINKGO LEAVES IN BROILER DIET
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant
Correspondence to: Fuliang Cao, College of Forestry, Nanjing Forestry University, No.159, Longpan Road, Nanjing 210037, P.R. China. Tel.: 0086-25-85427099, 0086-13913948118. Fax: 0086-25-85424121. E-mail address: [email protected]
Accepted for publication 30th January 2015
Abstract
1. The purpose of this study was to evaluate the effects of supplementing diets
with 3 types of fermented Ginkgo-leaves (FGL) on growth, antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks. A total of 300 d-old broilers were randomly allocated to 4 dietary treatments with 6
ip t
2.
replications of 10 birds each. Birds were fed on basal diets (Control) or basal diets
cr
3.
us
or their combined fermentation (CAF group), respectively, for a 42 d feeding trial.
AF and CAF supplementation improved body weight gain (22-42 d) and feed conversion
an
ratio (22-42 d and 1-42 d). Concentrations of serum α-tocopherol in CAF group, as well as
M
hepatic α-tocopherol in the 3 FGL groups were increased, while hepatic ROS levels were greatly decreased in group AF and CAF. Chickens in AF and CAF groups had decreased
ed
hepatic protein carbonyls and MDA, as well as jejunal and ileal protein carbonyls. The T-SOD activities and GSH of both jejunum and ileum of the CAF group were higher than the other
4.
ce pt
groups.
Duodenal and jejunal villous height of birds fed on the AF and CAF diets were increased,
while jejunal crypt depth was decreased. Furthermore, birds fed on AF and CAF supplemented diets had increased ileal Lactobacilli populations. Decreased ileal and caecal
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
supplemented with 0.5% FGL with Candida utilis (CF group), Aspergillus niger (AF group),
Escherichia coli and Salmonellas populations was found for the birds fed on CAF supplemented diets. 5. The present study may indicate that the improved feed efficiency and intestinal functions in the group supplemented with AF and CAF are directly connected with the improved antioxidant capacity and intestinal microbial ecology.
INTRODUCTION With the removal of antibiotic growth promoters from poultry diets in different areas of the world, it is of interest to investigate potential alternatives to maintain good growth
ip t
performance, intestinal health and microbial populations, particularly to control the growth of harmful bacteria. Natural medicinal products originating from fungi or herbs have been used
cr
us
reported that the potential of botanical sources and applications for improvement of growth performance, digestive function and microbial populations of the gastrointestinal tract in
an
broiler chickens (Dei et al., 2008; Giannenas et al., 2010; Leusink et al., 2010).
M
Ginkgo biloba L. (Family Ginkgoaceae) is one of the traditional herbs in China which has been used in some areas of the world. Leaves of Ginkgo biloba are well known for its
ed
high content of flavonoids. Chemically, the active constituents of Ginkgo biloba leafs are flavonoids (flavone glycosides, primarily composed of quercetin, kaempferol and
ce pt
isorhamnetin glycosides), polysaccharides (polymers of glucose, rhamnose, arabinose, mannose, galactose and xylose) and a terpenoids fraction (ginkolides A, B, C, J and bilobalides) (Kleijnen and Knipschild, 1992; van Beek and Montoro, 2009; Li et al., 2012). In the last few years, large-scale cultivation of ginkgo trees has been initiated for urban road
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
as feed supplements for centuries in ethnoveterinary medicine. Previous studies clearly
landscape or fruit production in China, so about 40000 tons are produced every year. Therefore, it is important to find out a way to utilise this herbal resource as feed ingredient and unveil its potential economic value in the feed industry. It was reported that the use of solid-state fermentation might improve the nutritive value of this botanical resource when it is fed to poultry (Chen et al., 2009).
Presently Aspergillus niger and Candida utilis are the major probiotic strains applied in broilers (Chen et al., 2009). A. niger is a fungus that has the capacity to produce enzymes such as hemicellulases, hydrolases, pectinases, protease, amylase, lipases, and tannases (Pinto et al., 2001; Mathivanan et al., 2006), and is one of the major probiotic strains applied in
ip t
broilers (Tannock, 2001; Dei et al., 2008). C. utilis belongs to Saccharomyces, and can
cr
al., (2008) and Miyake et al. (2005) found that fermentation not only alters the original
us
bioactivities of herbs, resulting in new treatment effects, but also enhances the original
an
treatment efficacy of active ingredients. Chen et al. (2009) reported that the beneficial effects of broilers fed on fermented feed was probably not through single-strain fermentation, but due
M
to the 2-stage combined fermentation process using Bacillus subtilis and Saccharomyces cerevisiae strains. To promote processing of ginkgo leaves, we have developed a process for
ed
A. niger fermentation wherein the functionality of this resource is preserved and enhanced.
ce pt
Our previous researches confirmed that the use of A. niger-fermented Ginkgo biloba leaves (FGL) had a positive influence on growth performance and lipid metabolism in broiler chicks (Cao et al., 2012; Zhang et al., 2012). In a recent study, Zhang et al. (2013) found that diets supplemented with 0.5% (in the starter phase) and 1.0% (in the grower phase) of A.
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
secrete protease, amylase, and lipase (Santoso et al., 2001; Koh et al., 2002). Studies of Dei et
niger-fermented-G. biloba leaves products had favourable influences on intestinal morphology, digestion and absorption without any adverse effects in broiler chickens. Dietary total flavonoids and polysaccharides were most likely the key compounds responsible for the health-improving effect of the fermentation products (Zhang et al., 2012). Despite these findings, there has been a dearth of information on the possible beneficial effect of FGL on layer hens. The aim of the present study was to compare the effects of Ginkgo leaves
processed with single strain (C. utilis or A. niger) or combined (C. utilis + A. niger) fermentation on growth performance, antioxidant capacity, intestinal morphology and microbial ecology of broiler chicks. MATERIALS AND METHODS
ip t
Culturing of Candida utilis and Aspergillus niger
cr
laboratory strain isolates obtained from the College of Chemical Engineering, Nanjing Forest
us
University, Nanjing, Jiangsu, P.R. China. The seed culture of C. utilis, containing (g/l)
an
glucose 20, peptone 10, and yeast extract 10 at pH 5.0, was prepared in a flask on a reciprocal shaker at 200 rpm at 30°C for 24 h. Medium for shake flask culture contains (g/l): glucose 30,
M
ammonium sulphate 8, KH2PO4 3 and anhydrous MgSO4 0.25. The final amount of C. utilis was 6-9×109 CFU/ml. A. niger was cultured by an agar plating technique using Sabouraud
ed
dextrose agar (Oxoid Ltd., Basingstoke, UK) and incubated at 24°C for 7 d. A. niger spores
ce pt
were harvested by tapping the top of the plate when turned upside down. Spore counts were determined using a haemocytometer according to the Fuchs-Rosenthal technique to be approximately 4.0 × 106 spores/ml, which were equivalent to 0.25 g. Preparation of fermented- ginkgo leaves sample
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
The Candida utilis (C. utilis, NFU-Y-186) and A. niger NL-1 used in this study was
Comminuted (2.0 -mm sieve) dried ginkgo leaves picked during the last third of September (ginkgo garden for leaf use, Nanjing Forestry University, Jiangsu Province, P.R. China) were used for this study. They were divided into 3 lots after autoclave sterilisation, and were fermented using yeast C. utilis, A. niger or their combination, respectively. The solid-state fermentation medium contained 10 g solid medium (ginkgo leaves : wheat bran : corncob = 8 : 1.5 : 0.5) and 16 ml nutritive salt (glucose : urea: (NH4)2SO4 :
peptone : KH2PO4: MgSO4·7H2O = 4 : 2 : 6 : 1 : 4 : 1) and was inoculated with 10% (ml/ml) of the C. utilis seed for an aerobic fermentation. 0.1% of the A. niger seed was inoculated into the fermentation medium for fermentation. Both samples were fermented for 48 h at 28°C-30°C. For the combined fermentation, medium was inoculated with 1 ml C. utilis seed
ip t
at 28°C-30°C for a 24 h aerobic fermentation in the first stage and then 2 ml A. niger seed was
cr
Six fermented samples were taken randomly, and were spread on a polythene sheet in a
us
room at 30-40°C, dried for 6 d up to about 900 g/kg of the dry matter, and ground to pass
an
through a 0.15-mm sieve. The changes of the ingredients before and after the fermentation are shown in Table 1. Repetitious examination showed that the proportion of components in
M
polysavone was constant within a minute range (variation coefficient
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks a
b
a
c
a
d
c
X. Zhang , Z. Sun , F. Cao , H. Ahmad , X. Yang , L. Zhao & T. Wang a
College of Forestry, Nanjing Forestry University, Nanjing, 210037, P.R. China,
b
Department of Animal Husbandry and Veterinary Science, Jiangsu Polytechnic College of Agriculture and Forestry c
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, P.R. China d
College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, Jiangsu Accepted author version posted online: 13 Apr 2015.
Click for updates To cite this article: X. Zhang, Z. Sun, F. Cao, H. Ahmad, X. Yang, L. Zhao & T. Wang (2015): Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks, British Poultry Science, DOI: 10.1080/00071668.2015.1030590 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1030590
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2015.1030590
CBPS-2014-359 MacLeod, March 2015
ip t
Ed. Kjaer, February 2015;
cr
us
capacity, intestinal morphology and microbial ecology in broiler chicks
X. ZHANG, Z. SUN1, F. CAO, H. AHMAD2, X. YANG, L. ZHAO3 AND T. WANG2
1
M
an
College of Forestry, Nanjing Forestry University, Nanjing, 210037, P.R. China,
Department of Animal Husbandry and Veterinary Science, Jiangsu Polytechnic College of
ed
Agriculture and Forestry, 2College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, P.R. China and 3College of Chemical Engineering, Nanjing
ce pt
Forestry University, Nanjing, 210037, Jiangsu
Running title: GINKGO LEAVES IN BROILER DIET
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
Effects of dietary supplementation with fermented Ginkgo leaves on antioxidant
Correspondence to: Fuliang Cao, College of Forestry, Nanjing Forestry University, No.159, Longpan Road, Nanjing 210037, P.R. China. Tel.: 0086-25-85427099, 0086-13913948118. Fax: 0086-25-85424121. E-mail address: [email protected]
Accepted for publication 30th January 2015
Abstract
1. The purpose of this study was to evaluate the effects of supplementing diets
with 3 types of fermented Ginkgo-leaves (FGL) on growth, antioxidant capacity, intestinal morphology and microbial ecology in broiler chicks. A total of 300 d-old broilers were randomly allocated to 4 dietary treatments with 6
ip t
2.
replications of 10 birds each. Birds were fed on basal diets (Control) or basal diets
cr
3.
us
or their combined fermentation (CAF group), respectively, for a 42 d feeding trial.
AF and CAF supplementation improved body weight gain (22-42 d) and feed conversion
an
ratio (22-42 d and 1-42 d). Concentrations of serum α-tocopherol in CAF group, as well as
M
hepatic α-tocopherol in the 3 FGL groups were increased, while hepatic ROS levels were greatly decreased in group AF and CAF. Chickens in AF and CAF groups had decreased
ed
hepatic protein carbonyls and MDA, as well as jejunal and ileal protein carbonyls. The T-SOD activities and GSH of both jejunum and ileum of the CAF group were higher than the other
4.
ce pt
groups.
Duodenal and jejunal villous height of birds fed on the AF and CAF diets were increased,
while jejunal crypt depth was decreased. Furthermore, birds fed on AF and CAF supplemented diets had increased ileal Lactobacilli populations. Decreased ileal and caecal
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
supplemented with 0.5% FGL with Candida utilis (CF group), Aspergillus niger (AF group),
Escherichia coli and Salmonellas populations was found for the birds fed on CAF supplemented diets. 5. The present study may indicate that the improved feed efficiency and intestinal functions in the group supplemented with AF and CAF are directly connected with the improved antioxidant capacity and intestinal microbial ecology.
INTRODUCTION With the removal of antibiotic growth promoters from poultry diets in different areas of the world, it is of interest to investigate potential alternatives to maintain good growth
ip t
performance, intestinal health and microbial populations, particularly to control the growth of harmful bacteria. Natural medicinal products originating from fungi or herbs have been used
cr
us
reported that the potential of botanical sources and applications for improvement of growth performance, digestive function and microbial populations of the gastrointestinal tract in
an
broiler chickens (Dei et al., 2008; Giannenas et al., 2010; Leusink et al., 2010).
M
Ginkgo biloba L. (Family Ginkgoaceae) is one of the traditional herbs in China which has been used in some areas of the world. Leaves of Ginkgo biloba are well known for its
ed
high content of flavonoids. Chemically, the active constituents of Ginkgo biloba leafs are flavonoids (flavone glycosides, primarily composed of quercetin, kaempferol and
ce pt
isorhamnetin glycosides), polysaccharides (polymers of glucose, rhamnose, arabinose, mannose, galactose and xylose) and a terpenoids fraction (ginkolides A, B, C, J and bilobalides) (Kleijnen and Knipschild, 1992; van Beek and Montoro, 2009; Li et al., 2012). In the last few years, large-scale cultivation of ginkgo trees has been initiated for urban road
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
as feed supplements for centuries in ethnoveterinary medicine. Previous studies clearly
landscape or fruit production in China, so about 40000 tons are produced every year. Therefore, it is important to find out a way to utilise this herbal resource as feed ingredient and unveil its potential economic value in the feed industry. It was reported that the use of solid-state fermentation might improve the nutritive value of this botanical resource when it is fed to poultry (Chen et al., 2009).
Presently Aspergillus niger and Candida utilis are the major probiotic strains applied in broilers (Chen et al., 2009). A. niger is a fungus that has the capacity to produce enzymes such as hemicellulases, hydrolases, pectinases, protease, amylase, lipases, and tannases (Pinto et al., 2001; Mathivanan et al., 2006), and is one of the major probiotic strains applied in
ip t
broilers (Tannock, 2001; Dei et al., 2008). C. utilis belongs to Saccharomyces, and can
cr
al., (2008) and Miyake et al. (2005) found that fermentation not only alters the original
us
bioactivities of herbs, resulting in new treatment effects, but also enhances the original
an
treatment efficacy of active ingredients. Chen et al. (2009) reported that the beneficial effects of broilers fed on fermented feed was probably not through single-strain fermentation, but due
M
to the 2-stage combined fermentation process using Bacillus subtilis and Saccharomyces cerevisiae strains. To promote processing of ginkgo leaves, we have developed a process for
ed
A. niger fermentation wherein the functionality of this resource is preserved and enhanced.
ce pt
Our previous researches confirmed that the use of A. niger-fermented Ginkgo biloba leaves (FGL) had a positive influence on growth performance and lipid metabolism in broiler chicks (Cao et al., 2012; Zhang et al., 2012). In a recent study, Zhang et al. (2013) found that diets supplemented with 0.5% (in the starter phase) and 1.0% (in the grower phase) of A.
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
secrete protease, amylase, and lipase (Santoso et al., 2001; Koh et al., 2002). Studies of Dei et
niger-fermented-G. biloba leaves products had favourable influences on intestinal morphology, digestion and absorption without any adverse effects in broiler chickens. Dietary total flavonoids and polysaccharides were most likely the key compounds responsible for the health-improving effect of the fermentation products (Zhang et al., 2012). Despite these findings, there has been a dearth of information on the possible beneficial effect of FGL on layer hens. The aim of the present study was to compare the effects of Ginkgo leaves
processed with single strain (C. utilis or A. niger) or combined (C. utilis + A. niger) fermentation on growth performance, antioxidant capacity, intestinal morphology and microbial ecology of broiler chicks. MATERIALS AND METHODS
ip t
Culturing of Candida utilis and Aspergillus niger
cr
laboratory strain isolates obtained from the College of Chemical Engineering, Nanjing Forest
us
University, Nanjing, Jiangsu, P.R. China. The seed culture of C. utilis, containing (g/l)
an
glucose 20, peptone 10, and yeast extract 10 at pH 5.0, was prepared in a flask on a reciprocal shaker at 200 rpm at 30°C for 24 h. Medium for shake flask culture contains (g/l): glucose 30,
M
ammonium sulphate 8, KH2PO4 3 and anhydrous MgSO4 0.25. The final amount of C. utilis was 6-9×109 CFU/ml. A. niger was cultured by an agar plating technique using Sabouraud
ed
dextrose agar (Oxoid Ltd., Basingstoke, UK) and incubated at 24°C for 7 d. A. niger spores
ce pt
were harvested by tapping the top of the plate when turned upside down. Spore counts were determined using a haemocytometer according to the Fuchs-Rosenthal technique to be approximately 4.0 × 106 spores/ml, which were equivalent to 0.25 g. Preparation of fermented- ginkgo leaves sample
Ac
Downloaded by [Nanyang Technological University] at 02:47 28 April 2015
The Candida utilis (C. utilis, NFU-Y-186) and A. niger NL-1 used in this study was
Comminuted (2.0 -mm sieve) dried ginkgo leaves picked during the last third of September (ginkgo garden for leaf use, Nanjing Forestry University, Jiangsu Province, P.R. China) were used for this study. They were divided into 3 lots after autoclave sterilisation, and were fermented using yeast C. utilis, A. niger or their combination, respectively. The solid-state fermentation medium contained 10 g solid medium (ginkgo leaves : wheat bran : corncob = 8 : 1.5 : 0.5) and 16 ml nutritive salt (glucose : urea: (NH4)2SO4 :
peptone : KH2PO4: MgSO4·7H2O = 4 : 2 : 6 : 1 : 4 : 1) and was inoculated with 10% (ml/ml) of the C. utilis seed for an aerobic fermentation. 0.1% of the A. niger seed was inoculated into the fermentation medium for fermentation. Both samples were fermented for 48 h at 28°C-30°C. For the combined fermentation, medium was inoculated with 1 ml C. utilis seed
ip t
at 28°C-30°C for a 24 h aerobic fermentation in the first stage and then 2 ml A. niger seed was
cr
Six fermented samples were taken randomly, and were spread on a polythene sheet in a
us
room at 30-40°C, dried for 6 d up to about 900 g/kg of the dry matter, and ground to pass
an
through a 0.15-mm sieve. The changes of the ingredients before and after the fermentation are shown in Table 1. Repetitious examination showed that the proportion of components in
M
polysavone was constant within a minute range (variation coefficient
