4.1. Components of sweet sorghum stem juice

Transcription

1 4.1. Components of sweet sorghum stem juice The stem juice of sweet sorghum is rich in fermentative sugar and is a desirable alcoholic fermentation material. It is difficult to measure the juice Sugar content in the process of production. The sugar content is commonly expressed with juice brix degree, but the relation between sugar content and brix degree has not been very cleared The objective of this study was to find out their relation and tell the sugar content by means of the measurement of juice saccharine more accurately, which may provide a theoretical basis for crop breeding and fermentation. In addition to fermentative sugar, other kinds of sugars are also found in the stem juice of sweet sorghum. The acquirement of the contents of different sugars is beneficial to the enhancement of alcohol production rate. There are also some ammonia acids and minerals in the juice, measuring their contents enables us to use sweet sorghum better with multi-purpose. The current research determined the sugar content and brix degree of different varieties in different growth stages. The results gave a scientific basis for the arrangement in the varieties and their sowing dates, so as to prolong the fermentation period and increase the efficiency of the alcoholic fermentation device usage. Therefore, this study has a practical instructive meaning for sweet sorghum breeding as well as the cultivation or fermentation, and the materials and methods as follows: A. Materials and Their Sources RIO US: recommended variety; Shennong No.2: Shenyang Agricultural University; 6AX1022: Liaoning Academy of Agricultural Science; Jitian 2: Jilin Academy of Agricultural Science; Longshi 1: Heilongjiang Academy of Agricultural Science; 6AXN249: Shenyang Agricultural University; B. Field Experiment a. Field planning Randomized blocks and triplication have been applied, among the triplication, the same material placed in different row. b. Plot area and density Plot area with 10 m long row, 0.7 m row distance, 5 ridges in one replication. The density of longshi 1 and Jitian2 are 8000 plants per Mu, others c. Seeding and fertilization Sowing in May 7. Base manure was spread 30t/ha, ammonia phosphate kg/ha and urea 300kg/ha.

2 d. Field management Appropriate intertilling, continuous operation, weeding by hand and intertill by machine. C. Measurement Methods At different period, two plants as a sample in each plot were randomly chosen for measuring their stem weights, and then juice of the stems were extracted by IJ-305 squeezer, and finally measuring glucose fructose sucrose content by high performance liquid chromatography, starch and total sugar content by another one spectrophotometry, Amino acid content by high speed amino acid analyzer, crude protein content by nitrogen and protein analyzer, total Phosphorus content by ammonia Vanadate and ammonia molybdenum spectrophotometry, mineral element content by atomic absorption spectrometry Brix degree in sweet Sorghum stem A. Changing Law of Brix Degree of juice in sweet sorghum stem The research shows that the brix degree of juice in sweet sorghum stem is lower before heading stage. After that, with the grain forming, the brix degree straightly increase towards its maximum at harvest stage. For whole Tab Brix Degree of different Varieties at different period variety time (D/M) 26/8 5/9 13/9 4/10 10/10 15/10 29/10 RIO Shennong No AX Jitian Longshi AXN Stem or each section of the stem, its brix degree rises as the plant growing it is the period within heading the first ten days and the third ten days that the degree increases distinctly. From Tab.4.1.1, it is known although among the six varieties, the brix degree changing law and the maximum degree period are not same, the present time of the maximum value is highly according to the harvest stage. Therefore, sweet sorghum should be harvested at the grain maturing stage in which both high sugar content and grain yield can be obtained. B. Analysis on Brix Degree of Juice in internodes and Whole Stem From Tab.4.1.2, it can find that the brix degrees are different in every internodes, the tendency is low-high-low from top to low position, and most

3 Tab internode brix degree of different varieties ( ) variety Rio Shennong No.2 6AX1022 Jitian 2 Longshi 1 6AXN249 internode Varieties highest brix degrees are occurred at 4 to 6th internodes from the top. In order to exactly measure the brix degree of stem, the juice for test should be extracted from whole stem. Varieties highest brix degrees are occurred at 4 to 6th internodes from the top. In order to exactly measure the brix degree of stem, the juice for test should be extracted from whole stem theoretically. However, it is impossible to extract the stalk juice on field. The purpose of this trial is to find a way of testing brix degree by one internode to present the one of whole stem, Through testing the brix degree of each internode of a stem (as shown in Tab ). it is known that the brix of some internodes can not present the one of whole stalk, because the brixes are different for each internode. The average brix degree for all internodes are suggested to be as the one of whole stalk. C. Comparison of Brix Degree of Different Varieties in the Same Period Variance analysis of test results in different three days indicates that the difference is significant between brix degrees of different varieties. The test shows that the brix degree of Longshi 1 is the highest and the one of 6AXN249 is the lowest. There RIO,

4 6AX1022, Shennong No.2 and Jitian 2. in the midst of them. Statistics analysis shows before September, the brix degree of Longshi 1 is the highest, but with the growing of plants, at the beginning of September, the difference becomes smaller until no any gap between them. Thus, for prolonging the period of ethanol production from sweet sorghum, the order of seeding time are Longshi 1, Shennong No.2. Tab Comparison between Internodal Mean Brix Degree (IMBD) and Whole Stalk Brix Degree (TBD) Variety Rio Shennong No.2 6AX1022 Jitian 2 Longshi 1 Time (D/M) 26/8 5/9 13/9 Plot q r s q r s q r s MBD TBD IMBD TBD IMBD TBD IMBD TBD IMBD TBD Analysis of Total Sugar Content in Juice of Sweet Sorghum There are plenty of sugar in the juice of sweet sorghum stem. However, how many kinds of sugars exist in the juice is still a question. Based on our results, it was not sufficient to regard the sum of sucrose, glucose and fructose as the total sugar content traditionally. The test by enthrone spectrophotometry shows the following kinds of sugars are existed in the juice of sweet sorghum, stem: xylose, ribose, arabinose, fructose, sorbose, galactose, mannose, sucrose glucose, polyglucose and glucoses. Of course, the total sugar content is much more than that of sucrose, glucose and fructose. Based on the test results as shown in Tab.4.1.4, the variance and the multiple comparison have proved both the variety and test time have a significant influence on sugar content. So in order to prolong the period of ethanol production and get more sugar, it is suggested that not only the varieties should be combined with but also the different harvest time should be planned Relationship between, Total Sugar Content and Brix Degree It has been doubted if the brix degree equals to sugar content. The experiment confirmed that the sugar content usually larger than brix degree. Variance analysis also indicates that sugar content (Y) of all varieties have a significant(!y) or extremely significant (!y!y) Line correlation with brix degree (X). Their line correlations can be respectively described as Tab

5 Tab Total sugar content of different variety in different period Variety Time (D/M) 4/10 10/10 15/10 25/10 29/10 Rio Shennong No AX Jitian Longshi AXN Tab Line Correlation between Sugar Content and Brix Degree Variety Equation R RIO Y= X 0.89 Shennong No.2 Y= X AX1022 Y= X 0.90 Jitian 2 Y= X 0.97 Longshi 1 Y= X AXN249 Y= X 0.97 Tab shows the sugar content with brix degree has a positive correlation. Based these equations, we can predicts the sugar content by measuring the brix degree easily. In order to use equation more convenient, we dealt with total sugar content (y) and brix degree (x). Table shows the mean Y and mean X of the 6 varieties at different periods. Variance analysis appears extremely Significant between Y and X, Our aim is this equation can be used in other varieties besides the tested ones. The line-correlation is: y= x Tab Mean total sugar content and mean brix degree time time (D/M) 4/10 10/10 15/10 25/10 29/10 brix degree total sugar content Contents of some main sugars

6 A. Glucose content Glucose is dextrose hose being present in all plant organs and tissues. Glucose has two crystals: one is? -glucose separated out from alcohol or water solution under room temperature, with melting point of 146 C and [? ] C; another is? - glucose separated out from hot pyridine solution of 148 to 150 C, [? ] C. D-glucose is the only form in natural world with a saccharinely of 0.69 times as much sucrose. Glucose is the primary material of plant photosynthesis. For C4 species, besides the Calvin cycle of glucose formation, there is also a four-carbon pathway for CO 2 fixation in mesophyll cells, therefore they have great potential for CO 2 assimilation. Sweet sorghum is a C4 crop, with lower CO 2 compensation point, higher light saturation point and weak photorespiration, and consequently has a higher biological yield. Glucose is a substrate of respiration and also a component of sucrose, starch and cellulose. As a reducing sugar, glucose can be fermented by saccharomycete. In fermentation, acetic aldehyde and CO 2 are produced through decarboxylation of pyruvic acid formed from the dehydrogenation of glucose, then acetic aldehyde is dehydrogenated and alcohol is produced. The whole process is under anaerobic and enzymatic conditions, which is known as alcohol-producing fermentation. Glucophosphate ester can be transformed into fructophosphate ester by isomerase. Glucose can form starch either with ADPG (or UDPG) as offering under amylosucurose, or with glucophosphate ester as offering under phosphatase transferring glucose to an introducer. Cellulose is formed through times of translocation of glucose unit to the glucose chain from GDPG under transferase. Tab Glucose content of different Varieties (%) 1989 Variety Time (D/M) 27/9 6/10 12/10 T* Rio Shennong No AX Jitian Longshi T: sampled on October 6 and measured on October 12 From Tab.4.1.7, samples were obtained for different varieties in different growth stages. The juice was extracted and was to be analyzed for glucose content. The

7 variance analysis showed that both varieties and growth stages affected glucose content very significantly. The multiple comparison results using SSR method showed that Jitian-2 was highest in glucose with difference from the lowest Rio; the glucose content of Jitian 2 had significant difference with that of Longshi 1 and 6AX1022, very significant with that of Shennong No.2 and Rio; there is no significant difference among 6AX1022, longshi 1, Shennong No.2 and Rio. As for the growth stages, October 6 sampling, then stored until October 12 for extraction and analysis showed highest glucose content, Very significantly different from other stages, while there is no significant difference among the latter. In summary, varieties were an very important factor determining the glucose content, there were differences in glucose among varieties; there were no significant differences in that among growth stages, but the stored sample after harvest gave obviously higher glucose content. B. Fructose content Fructose is a hexose with reductive character.? -pyranofructose is its free type, and? -furanofructose the combined type. The natural fructose is all levulose,? -Dfructose with [? ] C,? -D-fructose with [? ] C. Fructose is a colorless crystal with strong hygroscopicity. It is sweetest among the sugars, with a saccharinity of 1.15 to 1.5 times as much as sucrose, so it can be used as nutrient and as preserving agent. Fructose can be combined with saccharomycete. It is first transformed into fructophosphate ester, and then enter the EMP pathway. The sucrose synzyme existing in high plants can use uridine diphosphate glucose (UDPG) as the offering of glucose to form sucrose with fructose. It can also be synthesized under sucrose phosphate using UDPG as offering of glucose and 6-p-fructose as receptor. The first product of above reaction is phosphorescence that is then hydrolyzed to sucrose under phosphate. In plant photosynthetic tissue sucrose phosphoresce is more active, while in nonphotosynthetic tissue sucrose synzyme is mere active. Tab shows Fructose contents of 5 varieties, Rio.Shennong No.2, 6AX1022, Jitian 2, Longshi 1, in 4 growth stages, September 27, October 6, October 12 and October 6 sampling but October 12 measuring, were analyzed. The results indicated that varieties were not important determinant for fructose content. Of the 5 varieties, only Jitian 2 was significantly different in fructose content from Rio, whereas there was no big difference among others. However, growth stages affected fructose content very much. October 6 sampling but October 12 measuring got a highest fructose content, very significantly different from other stages among which no significant difference was found. Like glucose, fructose increased very significantly after storage. This means that storage exerts a large effect on fructose content. Tab Fructose content in stem juice (%) 1989 Variety Time (D/M)

8 27/9 6/10 12/10 T* Rio Shennong No AX Jitian Longshi T: Sampled on October 6 and measured on October 12 C. Sucrose content Sucrose is a widely existed disaccharide in natural world, and it is a non-reducing sugar. It is found in all photosynthetic plants, but is more in cane and beet so it is popularly called cane sugar on beet sugar sucrose plays an important role in plant physiology, it is not only the main product of photosynthesis, but also the main form of storage and accumulation of carbohydrate, sucrose is also the transportation form of carbohydrate within plant. Sucrose is disaccharide, when hydrolyzed, one molecule D-glucose and one molecule D-fructose are produced sucrose can be looked as a product of? -hydroxyglucoside and? -hydroxyfrucoside losing one molecule of water. The mixture of glucose and fructose formed from sucrose hydrolysis is defined as invert sugar. There is no hydroxyl group of glucoside in sucrose molecule, so sucrose cannot be changed to open chain structure. Therefore sucrose has no multiracial effect, can not form osazone. It has no reductive effect, is non-reducing sugar sucrose is dextrose with [?] 20 0=66.5 C. Saccharomycete can ferment sucrose. Tab Sucrose content in stem juice (%) 1989 Variety Time D/M) 27/9 6/10 12/10 T* Rio Shennong No AX Jitian Longshi T: Sampled on October 6. and measured on October 12 Tab shows the sucrose content of the tested varieties. It could the seen from the variance analysis and multiple comparison that varieties and growth stages had no significant effect on sucrose, and the content is relatively stable. Thus sucrose content does not greatly influence the fermented sugar and total sugar content.

9 D. Pooled analysis of glucose, fructose and sucrose From Table , Rio, Shennong No.2, 6AX1022, Jitian 2 and Longshi 1 were analyzed for sugar contents on September 27, October 6, October 12, and October 6 sampling but October 12 measuring. From Fig 1, we can know the differences among the 3 kinds of sugar contents. From the above 3 factors randomized block experiment, conclusions were made as follows: a. October 6 sampling and then stored until October 12 showed very significantly higher in sugar content than September 27, October 6 and October 12, indicating that storage could increase sugar content. The other 3 stages had no significant differences in sugar content, meaning that sugar content was stable during that period. b. sucrose content was very significantly different with glucose and fructose, while glucose content was not obviously different from fructose. This mean that sucrose prevailed over glucose and fructose in the stem sap of sweet sorghum. c. Sucrose, glucose and fructose contents showed great significant difference for Rio, Shennong No.2, 6AX1022 and Longshi 1, but no significant difference for Jitian 2. d. The glucose content in Jitian 2 varied significantly with those of the other 4 varieties. e. The fructose content showed no significant variance among the studied varieties. Tab Glucose, Fructose, sucrose content in stem juice of different varieties at different period (%) 1989 Time (D/M) 27/9 6/10 variety Glucose Fructose Sucrose Glucose Fructose Sucrose Rio shennong No AX Jitian Longshi Time (D/M) 12/10 T* variety Glucose Fructose Sucrose Glucose Fructose sucrose Rio shennong No ,

10 6AX Jitian Longshi * T: Sampled on October 6 and measured on October 12 Fig Glucose, Fructose and sucrose content in stem juice Starch content

11 Starch is a material of white and colorless, existing in granule forms of spherical or oval depending on plant types. It consists of amylose and amylopectin, and in most case the former accounts for 10 to 20% and the latter 80 to 90% It transfers into D- glucose by complete hydrolysis and into maltose by partial hydrolysis. The water solution of starch of dextrose with [? ] 20 0 =201.5 to 205 C. The mean specific weight is 1.5. Looking at the structure of starch, we know that at the end position of the polyglucoside chain there is free semi-acetalhydroxyl. But starch generally does not present reductive character, because there is only one semi-acetal hydroxyl in every hundreds or even thousands of glucose units. Tab Starch content of stem juice (%) 1990 Variety Time (D/M) 4 /10 10/10 15/10 25/10 29/10 Rio Shennong No AX Jitian Longshi AXN Starch is the main storing form of sugar and energy in plants existing with great amount in seeds, fruits, root and stem tubers, and with small amount in leaves and stems. Starch can not be fermented by saccharomycete directly, it needs to be dextrinized and saccharified, turning into fermentable, and then can be used to produce alcohol through fermentation. Starch content was measured for 6 varieties, Rio, Shennong No.2, 6AX1022, Jitian 2, Longshi 1 and 6AXN249, at 5 growth stages, October 4, October 10, October 15, October 25 and October 29 (Table 11). Variance analysis and multiple comparison results showed that varieties had little affect on starch content, among the 6 varieties only Shennong No.2 and 6AXN249 exhibited significant difference. Growth stages affected starch content greatly starch content on October 4 was highest, very significantly different from those on the other stages except October 10. starch content on October 10 had no significant difference with those on October 4 and October 15, but had significant difference with these on October 25 and October 29. There were no significant differences in starch content among October 15, October 25 and October 29. From the above analysis, we know that starch is very low in stem juice, it contributes in alcohol production with small amount so the stem juice of sweet sorghum is a good saccharine source. Therefore, the alcohol production with sweet sorghum stem

12 as raw material does not require complicated technology and expensive equipments, also the production period is short because of some procedures being left out. It is a low cost and easily operated alcohol producing method. As for starch content, early October is the best time for harvest when starch amount is high. starch can be utilized to produce alcohol through fermentation after saccharigation. Although starch content is not high, it is still important for a large production scale so the starch has also certain value in alcohol production Relation between total sugar and glucose, fructose and sucrose in stem juice According to the preceding discussion that the total sugar in the juice includes some penthouse, hexose and polysaccharide. of these sugars, glucose, fructose, sucrose and starch have examined in order to learn some internal regularities. Tab and Tab showed sugar contents of 5 varieties measured in The results in Table were measured on October 12 immediately after sampling, and those in Table were measured on October 12 after 6 days storage. From these tables we know that glucose, fructose and sucrose are the main parts of sugars in the juice, the sum of these 3 sugars is near to the total sugar amount but they are not the same thing, especially different sugars should not be simply summed up theoretically. So, the estimate of total sugar should be made by direct measurement or calculated according to the brix degree using the formula described earlier. Table Sugar Content in Stem Juice (%) Variety Rio Shennong No. 2 6AX1022 Jitian2 Longshi 1 total sugar content Glucose Fructose Sucrose Table Sugar Content in Stem Juice (%) 1989 Variety Rio Shennong No. 2 6AX1022 Jitian2 Longshi 1 total sugar content Glucose Fructose Sucrose It is known that the transformation from glucose to sucrose under enzyme is a simple process. Furthermore, sugar and starch contents are different among varieties, so considerations should be made on choosing higher glucose and fructose or higher sucrose. Perhaps it is better to select higher glucose and fructose sweet sorghum considering that the transformation from glucose to sucrose or starch, and

13 then starch and sucrose turning into glucose in the process of fermentation all consume energy.