CA2012278C - Mutant of microbacterium, a strain 851r, and a process for producing 851r nutrient solution by application of the strain - Google Patents
Mutant of microbacterium, a strain 851r, and a process for producing 851r nutrient solution by application of the strainInfo
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Abstract
This invention relates to one of microorganisms which is a mutant of Microbacterium named 851R. The main characteristics of this strain are as follows : a Gram-positive bacterium, motionless, with nitrate reduction ability, able to produce H2S, alive in milk heated to 63°C for 30 minutes or to 72°C for 15 minutes. The thallus is of red or orange red colour. However, the colour of the thallus is turned to pink at about 38°C
and to colourless at 40°C. In particular, the DNA of the strain contains R fragment which is detectable with a special R fragment DNA probe.
The invention also relates to an industrial process for producing single cell protein nutrient solution by an application of the bacterium. In this process, a Microbacterium strain 851R being cultivated in this invention is the producing strain, a soybean medium or starch medium acts mainly as the producing medium.
and to colourless at 40°C. In particular, the DNA of the strain contains R fragment which is detectable with a special R fragment DNA probe.
The invention also relates to an industrial process for producing single cell protein nutrient solution by an application of the bacterium. In this process, a Microbacterium strain 851R being cultivated in this invention is the producing strain, a soybean medium or starch medium acts mainly as the producing medium.
Description
A mutant of Microbacterium, A strain 851R, and a process for producing 851 nutrient solution by application of the strain The technical field of the invention This invention relates to a mutant of Microbacterium, particularly relates to a strain named 851R and an industrial fermentation process making use of the strain as a producing strain to produce single cell protein nutrient solution and a process for giving a nutrient solution.
The background of the invention Nowadays, many ways have been tried to select microorganisms which can be adopted in industrial fermentation to produce single cell protein easier to be ~ccimin~te~l by human body. The inventor of the present invention has made a method known to thepublic, which is a method to produce Se enriched single cell protein, vitamin E nutrient solution and bacteria manure with an ammonia resistance azotobacter. This methodoffers a cultivated azotobacter which possesses ammonia resistance and nitrogen fixing ability to make use of atmospheric nitrogen to produce single cell protein solution enriched with Zn, Se and vitamins by conventional industrial fermentation method.
The objective and summary of the invention The objective of this invention makes use of the strain 851R as a producing strain and is to provide conventional industrial fermentation method as a method to produce 851 nutrient solution by means of cultivating the strain 851R and designing proper media for the growth of the strain 851R, in which soybean acts as a main material.
Soybean is a kind of protein-rich food. Because soybean contains proteinase inhibitors, soybean protein is difficult to be totally assiminated by human body. However, after being assiminated by the bacterium of the strain 851R, soybean protein is converted to single cell protein nutrient solution which is not only a protein source easily to be assiminated by human body but also a kind of nutrient solution with the following effects making central nervous system healthy, organism immunologic enhancement, endocrine function regulation, antisenility, some kinds of cancer cell growth inhibition.
The microorganism related to this invention is a mutant of Microbacterium, named85 lR. The main characteristics of this strain are as follows : a Gram-positive bacterium, motionless, with nitrate reduction ability, able to produce H2S, alive in milk heated at 63~C for 30 minutes or at 72~C for 15 minutes. The thallus is of red or orange red colour. However, the colour of the thallus is turned to pink at about 38~C
and to colourless at 40~C. In particular, the DNA of this strain contains R fragment which is detectable with a special R fragment DNA probe.
The invention also relates to an industrial method which makes use of bacteria to produce single cell protein nutrient solution. In this method, the Microbacterium strain 85 lR cultivated in this invention acts as the producing strain, soybean medium or starch medium acts as the producing media.
Detailed description of the invention The microorganism related to this invention is a mutant of Microbacterium named Microbacterium 851R. The main characteristics are as follows: a Gram-positive rod, motionless, with glycerolraffinose assimination ability, nitrate reduction ability, H2S-producing ability, alive in milk heated at 63~C for 30 minutes or at 72~C for 15minutes. The thallus is of red or orange red colour, the colour of the thallus is turned to pale at 38~C and to colourless at 40~C. In particular, the DNA of the strain contains R fragment which is detectable with a special R fragment DNA probe.
A strain 851R, a mutant of Microbacterium, has the essential characteristics of the sample on deposit with American Type Culture Collection. 12301 Parklawn Drive, Rockville, Maryland 20852 USA, and assigned A.T.C.C. No.53981.
The strain of 851R possesses distinctive characteristics to be distinguished from other strains in the genus of Microbacterium. The application of the strain 851R as a producing strain and the application of the nitrogen source-containing media designed and used in this invention can produce a single cell protein solution enriched with peptides, a variety of trace elements and vitamins (listed in an analytical report of the culture solution). More than 20 amino acids are contained in the culture solution. 100 ml of this culture solution contain: 593-4172 mg of amino acids, 0.3-1.0 mg of vitamin E, 0.1-0.3 mg of vitamin B, 0.5-1.0 mg of vitamin PP, 1.0-5.0 ppm of Se, 10-20 ppm of Zn, 5.0-10 ppm of Mo, 5.0-10 ppm of Co, 5-15 ppm of Mn, and 3.0-10 ppm of Cu.
The media formulated and used in the invention by weight are as follows:
l. Soybean medium:
Soybean powder 5 - 10 %
or soybean milk (calculated by the weight of soybean) 5 - 20 %
or various bean cakes, sunflower seed cake 5 - 10 %
Yeast extract or yeast powder 0.02 - 0.5 %
CaCO3 0.05 - 0.5 %
K2HPO4 0.02 - 0.2 %
MgSO4 0.01 - 0.08 %
NaCl 0.01 - 0.08 %
Na2MoO4 5.0 - 50 ppm Na2SeO3 1.5 - 10 ppm ZnSO4 2.5 - 50 ppm CoCl2 2.5 - 20 ppm 2. Starch medium:
Fresh materials containing starch 10 - 20 %
(Sweet potato, potato etc.) or dry materials containing starch 1 - 10 %
(Corn powder etc.) Yeast extract 0.04 - 0.08 %
K2HPO4 0.05 - 0.1 %
MgSO4 0.02 - 0.04 %
CaCO3 0.05 - 0.5 %
NaCl 0.02 - 0.04 %
Na2MoO4 10 - 20 ppm Na2SeO3 1.5 - 20 ppm ZnSO4 5 - 20 ppm CoCl2 5 - 20 ppm Proper amounts of other trace elements necessary to human body may be added to the media mentioned above. The amounts of elements are in the range of 1.5 - 100 ppm.
A process for producing a nutrient solution by application of the strain 851R comprises the following steps: aeration culture is adopted in conventional tank fermenter installed with stirrer and gas transfer and when required, the fermenter can provide culture agitating to make cultures homogenized and can also provide culture aeration with filtered aseptic air to meet the oxygen need of bacterium and other microorganisms during culture period. The 851R strain being acting as a producing strain is propagated in the media formulated in this invention. After the three-step culture (the first, second and third seed culture), the thallus is inoculated into a preautoclaved producing medium and incubated for 36-64 hrs. During the culture period, aseptic air is passed through the medium, the aeration rate: 1:0.5 - 1.0 wm, stirring speed: 180 - 260 rpm, culture temperature: 28~C - 40~C. After the incubation is finished, the culture broth isautoclaved and harvested. Then the culture solution is directly packed to give products, also various series of products can be obtained by different processing methods to fulfill a variety of needs. Cola-type drinks may be prepared from the culture solution after precipitation and centrifugation at 15000 rpm, pressure (-0.2) -- (-0.4) kg/cm2. After concentration, the culture solution becomes concentrated nutrients. After being oven dried or spray dried, the culture solution is transformed into powder form and can be further processed to give capsules or tablets. For spray drying, the temperature at the entrance is 80~C-300~C, and 60~C-120~C at the exit. The culture solution can be added to wheat flour to bake bread and cake or other foods to replace water and eggs. The culture solution can also be used in the production of cosmetics.
The 851 nutrient solution possesses effects of anti-aging, improving constitutions, keeping liver and stomach in good condition. The following experiments give the details.
Experiment 1 Anti-lipid peroxidative effect Male Kunming mice, weighing 17.8 + 1.75 g, were used and divided into several groups according to body weight. Each group consisted of 10 mice. The tested mice were fed with 851 nutrient solution ( abbreviated to 851 NS ) instead of tap water for consecutive 13 days and fasted at 14th day. Then half of these tested mice were fed with tap water instead of 851 NS. Another half of the tested mice were continuously allowed to drink 851 nutrient, solution and ~lmini~tered with CCl4 intraperetoneally at 4 o'clock in the afternoon.
Mice of the control group were supplied with the same diet as the tested group, except tap water instead of 851 NS and administered with paraffin oil (dissolvent of CC14) intraperetoneally at 14th day.
Mice of CCl4 control group were supplied with the same diet and drunk tap water. The animals were fasted and administered with CC14 (1.87mmol/kg) intraperetoneally at afternoon.
At 15th day, the animals were decapitated and the liver sample were prepared. MDA
of the liver was detected according to the method of Hiroshi Ohakawa ( Hiroshi Ohakawa et al. Anal. Biochem. 95 :351-358, 1979 ), 1, 1, 3, 3, -tetramethoxy propane ( product of Japan TCI, E.P. grade ) was used as a standard sample for the detection of lipid peroxide, spectrophotometer was used to detect the absorbance of MDA. MDA
content is indicated as nmol/g liver. The data were analyzed statistically. The results were as follows:
group number of MDA nmolg liver p value mouse (H_SD) compared to compared to control group CCl4 group control 10 119.5 +27.2 - -CCl 10 1698.9+28.76 < 0.001 CCl +851 8 139.3+191.8 <0.001 The results indicated that 851 nutrient substantially solution possessed potent anti -lipid peroxidative activity in certain condition which might be useful for anti-aging.
Experiment 2 Swimming Test Male Kunming mice, weighing 15-24 g, were used and divided into several groups. The mice were fed with water, ginseng, Nestle milk powder and 851 NS, respectively.
Mice of 851 NS group were fed with 851 NS (5-6 ml/day) instead of tap water for 4 days.
Mice of control group were fed with tap water for 4 days.
Mice of Nestle milk powder group were fed with 0.01 % Nestle milk powder instead of tap water for 4 days.
Crude ginseng was minced into slices and immensed in boiling water for about 3 hrs.
and then boiled with gentle heat for 15 minutes twicely and prepared in 0.001 %
concentration to feed mice instead of tap water for 4 days. Swimming test was performed according to the method of Wang Yun-muo (Journal of Shanghai Medicine 2: 46-48, 1985). The results were shown as follows:
Group Water Crude ginseng Nestle milk powder 851NS
Number of 7 9 10 19 mouse Swimming time (second) 104.3+18.9 172+60.2 186.5+88.1 199.8+90.5 p value compared to - C 0.025 <0.05 <0.025 water group The results indicated that 851 nutrient solution significantly increased swimming time of mice and improved mices' physique.
Experiment 3 Inhibitory effect on gastric cancer cell (FGC) After discarding the used culture solution, gastric cancer cell (FGC), in good reproducing state, were digested with 0.02% EDTA and washed with 1640 medium containing 15 % bovine serium. Live cells were counted with tryphan blue and cultured in a total volume of 1 ml (lxlO5/ml) at 37~C for 24 hrs. Then the cells were washed and cultured in the same medium contain 851NS (0.2 ml/ml). Cells of the control group were cultured in a volume of 1 ml medium (10 ml of 1640 medium containing 15%
bovine serium and 2 ml of 851 NS) at 37~C for 24 hrs. The wells of each group were duplicated. Then the used medium was discarded. 0.9 ml 0.02% EDTA and 0.1 ml tryphan blue were added and live cells were counted.
inhibiting rate = _____________ X 100%
n, nl and n2 were he number of live cells in control group and tested group respectively.
The results indicated that the mean inhibition rate of 851 NS on the gastric cancer cells was 30%.
Experiment 4 Effect on Euplotes crassus Cell Division Euplotes crassus is a singular cell organism, which lives in seawater. The age of the cell is accouted by its asexual reproduction generation, ranged between 1000-1500.
Aging of Euplotes carssus cell is associated with some metabolic changes. Among these changes, the decrease of speed of cell division is one of the most significant characteristic of cell aging. The division speed of Euplotes carssus cell was significantly increased when the cells cultured in the seawater containing 10% 851 NS. The cell division speed (generation/day) of 5 days during the culture period was listed as follows:
~51 NS. Speed of cell division day 1 day 2 day 3 day 4 day 5 0 2.58 2.63 2.58 2.60 2.40 10% 4.00 3.70 2.86 4.13 4.00 The above table showed that the division speed of Euplotes crassus cell was increased 1.11-1.67 times when the seawater culture medium containing 10% 851 NS. Cell metabolism was improved when 851 NS was phagocytized into cells.
The division speed of the aged Euplotes crassus cell (aged 200 generation) was increased 1.4 times (from 0.33 to 0.79 generation/day) when culture medium containing 10% 851 nutrient solution.
The results indicated that 851 nutrient solution could improve the metabolism of the aged cells.
~xperiment 5 Effect of 851NS on body weight, haemoglobin and white cell classification of newly born mice 1. Materials:
Kunming mice were supplied by the animal center of Fujian Medical College. The batch number of 851 nutrient solution was 06.
2. Methods:
Twelve Kunming pregnant ice were fed with normal diet and tap water separately. After parturation, the 12 mice were divided into control and 851 NS group in random. The newborn mice were at a litter weighed when suckled. The female and newborn mice of control and 851 NS group were fed with normal diet or the diet containing 851 NS, respectively, during the period of suckling and 15 days after weaning. Then the newborn ice were weighed at a litter. The blood sample was taken from the eye ball of the newborn mice. The increase of the body weight, and classification of white cells of newborn mice were shown as follows:
Results and discussion l. Results:
l. 1 Table 1 showed body weight changes of newborn mice.
Table l Effect of 851NS on body weight increase of the newborn mice brood numbernewborn mice body weight p value*
number increase of each newborn mouse (g. X+ SD) control 4 34 14.20+0.75 851 4 34 14.36+1.29 < 0.05 * Compared with control group 1.2 Table 2 showed Effect of 851NS on white cell classification of newborn mice Table 2 (% X + D.SD) group micemorphonaclear p 1 valuelymphocyte p 2 value*
numberleukocyte (% X +SD) (% X _SD) control 34 58+10 42+9 851 34 42+12 <0.001 57+12 <0.001 * Compared with control group 2. Discussion 851 nutrient solution was a bioengineering product which contained more than 20 kinds of amino acids, several kinds of vitamins and trace elements. The results showed that 851 nutrient solution significantly increased the value of lymphocoyte of the newborn mice, which indicated that 851 NS could stimulate and improve the blood manufacturing function of the newborn mice.
The results also showed that 851NS could increase the body weight of the newbornmice, which indicated that some components of 851NS were nutritious for the newborn mice.
~xperiment 6 Therapeutic effect of 851NS on the subchronic injury to gastric mucosa in mice 1. Materials Kunming mice of both sex weighing 42-44 g were supplied by the animal center of Fujian Medical College. 851NS, 4N HCI and 0.1% indomethacin were used.
2. Methods 2.1 The establishment of the model of subchronic injury to gastric mucasain mice Eight mice were divided into 2 groups in random. Mice of the control group were administered with normal saline once daily, P.O. Mice of the tested group were administered with 4N HCl or 0.1% indomethacin alternately once daily, (P.O.) for two days. These mice were fed with normal diet and tap water. Ten days later, the mice were decapitated. The stomach was removed and washed with water. The gastric mucosa of the control group showed lustrous and red, while the gastric mucosa of the tested group were lustreless, pale and swelling. The gastric cavity was fill with 10%
neutral formalin for fixing, cut into sections with paraffin wax and stained with HE
staining. Gastric mucosa were examined with microscope and showed that the endothilium of the mucosa became thinner and covered with eroded keratinic substances, gastric gland scattered with eroded necrosis. No significant changes was observed in control group. It was confirmed that the method described above could become a model of subchronic injury.
2.2 Therapeutic effect of 851NS on mice subchronic injury of gastric moucosa Thirty mice were divided into control group and 851 group in random and administered with HCl or indomethacin alternative according to the method described above for 10 days. The mice of these two groups were fed with normal diet or the diet containing 851, respectively during 10 days and continued for 7 days after the ~11mini~tration of HCl or indomethacin was stopped. Then the mice wére decapitated. The change of the gastric moucosa was examined according to the method described above.
Result and Discussion There was no significant difference of the morphology of gastric moucosa between the control and 851 groups. The mucosa in the front part of the stomach of the 851 group was a little thicker than that of the control group. The results indicated that 851 was beneficial to the recovery of injury of gastric mucosa.
Experiment 7 Therapeutic effect of 851 on hemmorhage of gastric mucosa in rats Materials and Methods 1. Methods Rats were divided into control and 851 groups in random and fed with normal diet and the diet containing 851 (8 ml/rat), respectively. All rats were supplied with sufficient tap water. 15 days later, all rats were fasted for 14 hrs. and administed with indomethacin (2.5 mg/rat) subcutaneously once daily for two days. 18 hours after the second injection, rats were decapitated and the stomach was examined.
Results and Discussion 1 . Results Number and degree of acute hemorrhage of gastric mucosa: The hemorrhage of gastric mucosa in control 6 mice and 4 mice in 851 group was locally restricted as spotted, stripped or stretched situation. Diffused hemorrhage of gastric mucosa occurred in one rat of the control group. There were 6-12 spots hemorrhage of gastric mucosa in control group and 2-8 in 851 group. The number of hemorrhage of gastric mucosa of control group was significantly severe than that of 851 group.
2. Discussion The results indicated that indomethasin induced hemorrhage of gastric mucosa wasprevented when rats were fed with 851NS for 15 days.
Experiment 8 Effect of 851 NS on CCl4 induced subchronic hepatic injury in rat Material and Methods 1. Materials Twenty two Wistar rats (male 10 and female 12), weighing 140-220 g were supplied by the animal center of Fujian Medical College. CCl4 analytical grade, was diluted in purified peanut oil to the concentration of 40%. 851 NS, agar gel (product of Sh~ngh~i Chemical Agent Manufactory) and LEB multiple usage of electrophoresis equipment were used.
2. Methods Twenty two rats were divided randomly into 3 groups, i.e. control group (n=6), CCl4 group (n=8) and CCl4 + 851 group (n=8). Rats of CCl4 and CCl4 + 851 group were administered with CCl4 (0.1 ml/lOOg) subcutaneously twice a week (3 days interval), for 5 weeks. Rats of control and CCl4 groups were fed with normal diet. Rats of CCl4 +
851 group were fed with the diet containing 851 NS (8 ml/rat.day). All rats weresupplied with sufficient tap water and fed for 5 weeks. Five days after the lastadministration of CC14, blood was taken from the axillary vein. Serum was collected and liver function was detected. Electrophoresis of serum protein was performed with LEB electrophoresis equipment. Pathological changes of the liver were examined.
Results and Discussion 1. Results:
l . l Rat SGPT
Effect of 851 NS on CCl4 induced SGPT changes in rats group number SGPT(%,X + SD) p l* p 2*
control 6 29.8 + 8.0 CCl4 8 41.6 + 5.3 < 0.01 CCl4 + 851 8 31.0 + 5.5 > 0.05 <0.01 * pl compared with control group, p2 compared with CCl4 group.
1.2 There were no significant differences of thymol turbidity test (l-l l) and zinc sulfate turbidity test (Zn TT) between these groups.
1.3 Observation of liver by naked eye: The color and lustre of the liver capsule in control group were normal. The liver capsule was lustrous. The rat liver capsules of CCl4 group were pale brown and lustrous in accordance with the morphological changes of liver with watery degeneration. The rat liver mophology of CCl4 + 851 group was better than that of CCl4 group and poor than that of control group.
1.4 Observation of liver by microscope: In control group the liver histological structure of 5 rats were normal except one with small watery degeneration. Eight rats of CCl4 group showed significant liver watery degeneration. Cell plasma of the outer part of liver lobule showed diffused or focal reticulated areosis. Some cells in the central part of the liver loblue were normal and some showed degeneration. Fatty degenerated liver cells were scattered or focused and difficult to be differentiated from watery degenerated cells. Six rat liver of the CCl4 + 851 group showed less watery degeneration than that of CCl4 group, another 2 rats showed the same watery degeneration as that of CCl4 group. Rat liver histological changes were in accordance with the results of liver function SGPT. This indicated that the results of rat SGPT in all groups were reliable.
1.5 Electrophoresis of rat serum protein: Two serum samples of each group were used for electrophoresis. The results showed that there were no differences among these groups. These results indicated that CCl4 induced liver injury did still not cause changes of rat serum protein.
1.6 Increase of body weight: The increase of rat body weight of CCl4 + 851 groupwere more than that of the other groups, but there were not statistically significant differences among these groups (p~0.05).
2. Discussion The results of rat serum SGPT and morphological changes investigated by naked eye and microscope indicated that 851 showed preventive and therapeutic effects against CCl4 induced subchronic liver injury.
~xperiment 9 Effect of 851 NS on the body weight development of newborn and suckling mice Materials and Methods l. Materials Kunming mice were supplied by the animal center of Fujian Medial College. 851 NS(batch number 06) was used.
2. Methods 27 female Kunming mice after mating with the same species of male mice, when they were preliminary considered to be pregnant (with vaginal thrombus), were divided into control group and 851 groups randomly. Mice of control group were fed with normal diet while mice of the 851 group were fed with the diet containing 851. All mice were supplied with tap water. Seven mice were taken from each group in random and fedwith the same method described above separately after they were confirmed to be pregnant from the enlarging abdomen. Newborn mice per litter were weighed at the day of parturition, and after the period of 20 days suckling. The body weight and the external appearance of the newborn mice and suckling mice were investigated.
Results and Discussion 1. Results 1.1Effect of 851NS on the body weight of the newborn mice (just after parturition) Table 3Effect of 851 on the body weight of newborn mice group litter number number of body weight of average body p*
newborn mice each newborn weight of each per litter mice in the newborn mice same litter (g.X) in the group (g.x_50) Control l 13 1.54 2 lO 1.70 3 9 1.70 1.61 + 0.067 4 10 1.60 1.65 6 10 1.57 7 10 1.54 851 l 12 1.66 2 ll 1.77 3 8 2.15 4 9 1.85 1.83+0.15 <0.01 8 1.81 6 10 1.78 7 13 1.78 * Compared with control group.
1.2 Effect of 851NS on the body weight of suckling mice were shown in table 4.
Table 4 Effect of 851NS on the body weight of suckling mice group litter number of body weight of average of body weight p*
number suckling the suckling mice of the suckling mice in mice in the same litter the same group (g.X+50) (g.X) control 1 10 10.60 2 9 11.30 3 11 8.45 4 8 12.60 10.52+ 1.68 8 11.20 6 13 8.00 7 8 11.50 851 1 9 13.00 2 9 12.85 3 9 11.53 4 8 14.75 12.44 + 1.81 <0.05 8 14.36 6 10 10.64 7 13 9.98 * Compared with control group.
1.3 No abnormal phenomenon of external appearance, capacity for eating and general activities were observed.
2. Discussion 2.1 Table 3 showed that 851NS could promote the body weight development of the mouse embryo when the female mice were fed with 851 NS during the period of pregnancy (p < 0.01).
2.2 Table 4 showed that 851 NS could promote the body weight development of the suckling mice when the female mice were fed with 851 NS during the period of thesuckling (p < 0.05).
2.3 The results indicated that no abnormal development (such as fetus malformation) and adverse effect were observed when female mice were fed with 851 NS during 40 days period from pregnancy to suckling.
The embodiments of the present invention are described, but not limited, in detail as follows:
Example 1 A 2.5 ton tank fermenter was used in this production. The seed fermenter was of 0.5 ton. The amount of raw materials taken in was 40%, a 5% soybean medium was adopted as the producing medium containing: 5% of soybean powder, 0.04% of yeastextract, 0.05 % KH2PO4, 0.02 % of MgSO4, 0.02 % of CaC03, 0.02 % of NaCl, 10 ppmof Na2MoO4, 2.5 ppm of Na2SeO3, 10 ppm of ZnSO4, 2.5 ppm of CoCl2 and water.
The amount of inoculum was 1 %, the seed fermenter was of 2 liters, the cell age was 24 hrs., ( the first seed culture -- the second seed culture -- the third seed culture, the culture incubated for 24 hrs. was used as inoculum.) culture te",pel~ture was 30~C, stirring speed was 260 rpm, the aeration rate was l: 0.8 wm. The culture incubation lasted for 45 hrs. After terminating the incubation, the culture was autoclaved and packed into bottles through an assembly line, then autoclaved again to give final products. 100 ml of the culture solution contained: 3.7% of dry material, 2.06% of proteins, 0.23% of lipids, 0.75% carbohydrates, vitamins and mineral salts. (listed in Table 5) Table 5 Analytical report of 851 culture solution Constituents Unit Amount ConstituentsUnit Amount eatable part g 100 asparic acid mg 87.8 water g 96.3 gulutamic acidmg 93.8 protein g 2.06 serine mg 28.1 lipid g 0.23 proline mg 29.0 carbohydrate g 0.75 glycine mg 33.6 heat of capacitykcal 13.3 alanine mg 48.1 eatable threonine mg 30.0 cellulose g 0.26 valine mg 37.0 ash g 0.40 methionine mg 10.9 isoleucine mg 26.1 K mg 49.0 leucine mg 51.4 Na mg 18.0 tyrosine mg 22.0 Ca mg 39.5 phenylalaninemg 24.0 Fe mg 2.3 lysine mg 34.7 Zn mg 4.4 tryptophen mg 10.0 Cu mg 0.05 histidine mg 14.2 Mn mg 0.14 arginine mg 39.6 Mg mg 5.3 P mg 42.9 myristic acid %
Se mg - 0.05 palmitic acid % 14.4 stearic acid % 85.5 vitamin E mg 0.64 vitamin B2 mg 0.13 Vitamin PP mg 0.47 Example 2 An 8 ton tank fermenter was used in this production. The amount taken in of raw materials was 40%. It means that the amount of medium was 3.2 ton. A 10% soybeanmilk medium was adopted. The medium contained: 320 kg of soybeans, 1.28 kg of yeast extract, 1.6 kg of KH2PO4, 640 g of MgSO4, 640 g of CaC03, 640 g of NaCl, 32 g of Na2MoO4, 8 g of Na2SeO3, 32 g of ZnSO4, 8 g of CoCl2 and water. The amount of inoculum was 1 %, the cell age was 24 hrs., (the first seed culture, the second seed culture and the third seed culture, the culture incubated for 24 hrs. was used as inoculum.) The culture temperature: 30~C, the stirring speed: 220 rpm, the aeration rate: 1:0.8 wm. After fini.~hing the incubation in a seed fermenter, the inoculum was inoculated into an 8 ton tank fermenter, the culture temperature: 30~C, the stirring speed: 220 rpm, the aeration rate: 1:0.8 vvm. The incubation was terminated after 48 hrs. and the culture solution was autoclaved and packed into bottles through an assembly line, then autoclaved again to give the final products. 100 ml of the culture solution contained: aspartic acid 543 mg, glutamic acid 710 mg, serine 160 mg, glycine 280 mg, histidine 65 mg, argining 226 mg, threonine 232 mg, alanine 359 mg, proline 126 mg, tyrosine 101 mg, valine 283 mg, methionine 53 mg, isoleucine 262 mg, leucine 309 mg, phenylalanine 148 mg, lysine 212 mg, cystine 38 mg, the total amount: 4127 mg.
Example 3 The nutrient solution produced by the method given in example 2 was dried by a spray dryer usually used to make dried milk to give 851 nutrient powder. The temperature was of 80~C at the spray dryer entrance and 60~C at the exit. 0.8 ton of 851 nutrient solution gave 20.4 kg of dried powder, which was further processed into capsules. One capsule contained 0.28 g of dried powder or formulated with 80 kg of starch and 10 kg of honey to make granule preparations.
Example 4 The 851 nutrient solution produced by conventional fermentation method was centrifuged to get rid of precipitate, at 15000 rpm. Thus 1 ton of 851 nutrient solution gave 960 kg of supernatant. 851 fruit juice cola was prepared by adding 500 kg of fruit juice, 851 honey cola was prepared by adding 40 kg of honey. The supernatant mentioned above might also be flavoured to give a variety of 851 drinks with different taste according to needs.
Example S
The 851 nutrient solution produced by conventional fermentation method was centrifuged. The supernatant was concentrated by a film evaporator or a vaccum rotatory evaporator to five 851 concentrated nutrients. After evaporation, 0.5 ton of the supernatant gave 100 kg of concentrated nutrient. After adding 10 kg of honey, the concentrated nutrient was packed into small bottles with easy-to-open cap. This nutrient is convenient to be taken orally.
The background of the invention Nowadays, many ways have been tried to select microorganisms which can be adopted in industrial fermentation to produce single cell protein easier to be ~ccimin~te~l by human body. The inventor of the present invention has made a method known to thepublic, which is a method to produce Se enriched single cell protein, vitamin E nutrient solution and bacteria manure with an ammonia resistance azotobacter. This methodoffers a cultivated azotobacter which possesses ammonia resistance and nitrogen fixing ability to make use of atmospheric nitrogen to produce single cell protein solution enriched with Zn, Se and vitamins by conventional industrial fermentation method.
The objective and summary of the invention The objective of this invention makes use of the strain 851R as a producing strain and is to provide conventional industrial fermentation method as a method to produce 851 nutrient solution by means of cultivating the strain 851R and designing proper media for the growth of the strain 851R, in which soybean acts as a main material.
Soybean is a kind of protein-rich food. Because soybean contains proteinase inhibitors, soybean protein is difficult to be totally assiminated by human body. However, after being assiminated by the bacterium of the strain 851R, soybean protein is converted to single cell protein nutrient solution which is not only a protein source easily to be assiminated by human body but also a kind of nutrient solution with the following effects making central nervous system healthy, organism immunologic enhancement, endocrine function regulation, antisenility, some kinds of cancer cell growth inhibition.
The microorganism related to this invention is a mutant of Microbacterium, named85 lR. The main characteristics of this strain are as follows : a Gram-positive bacterium, motionless, with nitrate reduction ability, able to produce H2S, alive in milk heated at 63~C for 30 minutes or at 72~C for 15 minutes. The thallus is of red or orange red colour. However, the colour of the thallus is turned to pink at about 38~C
and to colourless at 40~C. In particular, the DNA of this strain contains R fragment which is detectable with a special R fragment DNA probe.
The invention also relates to an industrial method which makes use of bacteria to produce single cell protein nutrient solution. In this method, the Microbacterium strain 85 lR cultivated in this invention acts as the producing strain, soybean medium or starch medium acts as the producing media.
Detailed description of the invention The microorganism related to this invention is a mutant of Microbacterium named Microbacterium 851R. The main characteristics are as follows: a Gram-positive rod, motionless, with glycerolraffinose assimination ability, nitrate reduction ability, H2S-producing ability, alive in milk heated at 63~C for 30 minutes or at 72~C for 15minutes. The thallus is of red or orange red colour, the colour of the thallus is turned to pale at 38~C and to colourless at 40~C. In particular, the DNA of the strain contains R fragment which is detectable with a special R fragment DNA probe.
A strain 851R, a mutant of Microbacterium, has the essential characteristics of the sample on deposit with American Type Culture Collection. 12301 Parklawn Drive, Rockville, Maryland 20852 USA, and assigned A.T.C.C. No.53981.
The strain of 851R possesses distinctive characteristics to be distinguished from other strains in the genus of Microbacterium. The application of the strain 851R as a producing strain and the application of the nitrogen source-containing media designed and used in this invention can produce a single cell protein solution enriched with peptides, a variety of trace elements and vitamins (listed in an analytical report of the culture solution). More than 20 amino acids are contained in the culture solution. 100 ml of this culture solution contain: 593-4172 mg of amino acids, 0.3-1.0 mg of vitamin E, 0.1-0.3 mg of vitamin B, 0.5-1.0 mg of vitamin PP, 1.0-5.0 ppm of Se, 10-20 ppm of Zn, 5.0-10 ppm of Mo, 5.0-10 ppm of Co, 5-15 ppm of Mn, and 3.0-10 ppm of Cu.
The media formulated and used in the invention by weight are as follows:
l. Soybean medium:
Soybean powder 5 - 10 %
or soybean milk (calculated by the weight of soybean) 5 - 20 %
or various bean cakes, sunflower seed cake 5 - 10 %
Yeast extract or yeast powder 0.02 - 0.5 %
CaCO3 0.05 - 0.5 %
K2HPO4 0.02 - 0.2 %
MgSO4 0.01 - 0.08 %
NaCl 0.01 - 0.08 %
Na2MoO4 5.0 - 50 ppm Na2SeO3 1.5 - 10 ppm ZnSO4 2.5 - 50 ppm CoCl2 2.5 - 20 ppm 2. Starch medium:
Fresh materials containing starch 10 - 20 %
(Sweet potato, potato etc.) or dry materials containing starch 1 - 10 %
(Corn powder etc.) Yeast extract 0.04 - 0.08 %
K2HPO4 0.05 - 0.1 %
MgSO4 0.02 - 0.04 %
CaCO3 0.05 - 0.5 %
NaCl 0.02 - 0.04 %
Na2MoO4 10 - 20 ppm Na2SeO3 1.5 - 20 ppm ZnSO4 5 - 20 ppm CoCl2 5 - 20 ppm Proper amounts of other trace elements necessary to human body may be added to the media mentioned above. The amounts of elements are in the range of 1.5 - 100 ppm.
A process for producing a nutrient solution by application of the strain 851R comprises the following steps: aeration culture is adopted in conventional tank fermenter installed with stirrer and gas transfer and when required, the fermenter can provide culture agitating to make cultures homogenized and can also provide culture aeration with filtered aseptic air to meet the oxygen need of bacterium and other microorganisms during culture period. The 851R strain being acting as a producing strain is propagated in the media formulated in this invention. After the three-step culture (the first, second and third seed culture), the thallus is inoculated into a preautoclaved producing medium and incubated for 36-64 hrs. During the culture period, aseptic air is passed through the medium, the aeration rate: 1:0.5 - 1.0 wm, stirring speed: 180 - 260 rpm, culture temperature: 28~C - 40~C. After the incubation is finished, the culture broth isautoclaved and harvested. Then the culture solution is directly packed to give products, also various series of products can be obtained by different processing methods to fulfill a variety of needs. Cola-type drinks may be prepared from the culture solution after precipitation and centrifugation at 15000 rpm, pressure (-0.2) -- (-0.4) kg/cm2. After concentration, the culture solution becomes concentrated nutrients. After being oven dried or spray dried, the culture solution is transformed into powder form and can be further processed to give capsules or tablets. For spray drying, the temperature at the entrance is 80~C-300~C, and 60~C-120~C at the exit. The culture solution can be added to wheat flour to bake bread and cake or other foods to replace water and eggs. The culture solution can also be used in the production of cosmetics.
The 851 nutrient solution possesses effects of anti-aging, improving constitutions, keeping liver and stomach in good condition. The following experiments give the details.
Experiment 1 Anti-lipid peroxidative effect Male Kunming mice, weighing 17.8 + 1.75 g, were used and divided into several groups according to body weight. Each group consisted of 10 mice. The tested mice were fed with 851 nutrient solution ( abbreviated to 851 NS ) instead of tap water for consecutive 13 days and fasted at 14th day. Then half of these tested mice were fed with tap water instead of 851 NS. Another half of the tested mice were continuously allowed to drink 851 nutrient, solution and ~lmini~tered with CCl4 intraperetoneally at 4 o'clock in the afternoon.
Mice of the control group were supplied with the same diet as the tested group, except tap water instead of 851 NS and administered with paraffin oil (dissolvent of CC14) intraperetoneally at 14th day.
Mice of CCl4 control group were supplied with the same diet and drunk tap water. The animals were fasted and administered with CC14 (1.87mmol/kg) intraperetoneally at afternoon.
At 15th day, the animals were decapitated and the liver sample were prepared. MDA
of the liver was detected according to the method of Hiroshi Ohakawa ( Hiroshi Ohakawa et al. Anal. Biochem. 95 :351-358, 1979 ), 1, 1, 3, 3, -tetramethoxy propane ( product of Japan TCI, E.P. grade ) was used as a standard sample for the detection of lipid peroxide, spectrophotometer was used to detect the absorbance of MDA. MDA
content is indicated as nmol/g liver. The data were analyzed statistically. The results were as follows:
group number of MDA nmolg liver p value mouse (H_SD) compared to compared to control group CCl4 group control 10 119.5 +27.2 - -CCl 10 1698.9+28.76 < 0.001 CCl +851 8 139.3+191.8 <0.001 The results indicated that 851 nutrient substantially solution possessed potent anti -lipid peroxidative activity in certain condition which might be useful for anti-aging.
Experiment 2 Swimming Test Male Kunming mice, weighing 15-24 g, were used and divided into several groups. The mice were fed with water, ginseng, Nestle milk powder and 851 NS, respectively.
Mice of 851 NS group were fed with 851 NS (5-6 ml/day) instead of tap water for 4 days.
Mice of control group were fed with tap water for 4 days.
Mice of Nestle milk powder group were fed with 0.01 % Nestle milk powder instead of tap water for 4 days.
Crude ginseng was minced into slices and immensed in boiling water for about 3 hrs.
and then boiled with gentle heat for 15 minutes twicely and prepared in 0.001 %
concentration to feed mice instead of tap water for 4 days. Swimming test was performed according to the method of Wang Yun-muo (Journal of Shanghai Medicine 2: 46-48, 1985). The results were shown as follows:
Group Water Crude ginseng Nestle milk powder 851NS
Number of 7 9 10 19 mouse Swimming time (second) 104.3+18.9 172+60.2 186.5+88.1 199.8+90.5 p value compared to - C 0.025 <0.05 <0.025 water group The results indicated that 851 nutrient solution significantly increased swimming time of mice and improved mices' physique.
Experiment 3 Inhibitory effect on gastric cancer cell (FGC) After discarding the used culture solution, gastric cancer cell (FGC), in good reproducing state, were digested with 0.02% EDTA and washed with 1640 medium containing 15 % bovine serium. Live cells were counted with tryphan blue and cultured in a total volume of 1 ml (lxlO5/ml) at 37~C for 24 hrs. Then the cells were washed and cultured in the same medium contain 851NS (0.2 ml/ml). Cells of the control group were cultured in a volume of 1 ml medium (10 ml of 1640 medium containing 15%
bovine serium and 2 ml of 851 NS) at 37~C for 24 hrs. The wells of each group were duplicated. Then the used medium was discarded. 0.9 ml 0.02% EDTA and 0.1 ml tryphan blue were added and live cells were counted.
inhibiting rate = _____________ X 100%
n, nl and n2 were he number of live cells in control group and tested group respectively.
The results indicated that the mean inhibition rate of 851 NS on the gastric cancer cells was 30%.
Experiment 4 Effect on Euplotes crassus Cell Division Euplotes crassus is a singular cell organism, which lives in seawater. The age of the cell is accouted by its asexual reproduction generation, ranged between 1000-1500.
Aging of Euplotes carssus cell is associated with some metabolic changes. Among these changes, the decrease of speed of cell division is one of the most significant characteristic of cell aging. The division speed of Euplotes carssus cell was significantly increased when the cells cultured in the seawater containing 10% 851 NS. The cell division speed (generation/day) of 5 days during the culture period was listed as follows:
~51 NS. Speed of cell division day 1 day 2 day 3 day 4 day 5 0 2.58 2.63 2.58 2.60 2.40 10% 4.00 3.70 2.86 4.13 4.00 The above table showed that the division speed of Euplotes crassus cell was increased 1.11-1.67 times when the seawater culture medium containing 10% 851 NS. Cell metabolism was improved when 851 NS was phagocytized into cells.
The division speed of the aged Euplotes crassus cell (aged 200 generation) was increased 1.4 times (from 0.33 to 0.79 generation/day) when culture medium containing 10% 851 nutrient solution.
The results indicated that 851 nutrient solution could improve the metabolism of the aged cells.
~xperiment 5 Effect of 851NS on body weight, haemoglobin and white cell classification of newly born mice 1. Materials:
Kunming mice were supplied by the animal center of Fujian Medical College. The batch number of 851 nutrient solution was 06.
2. Methods:
Twelve Kunming pregnant ice were fed with normal diet and tap water separately. After parturation, the 12 mice were divided into control and 851 NS group in random. The newborn mice were at a litter weighed when suckled. The female and newborn mice of control and 851 NS group were fed with normal diet or the diet containing 851 NS, respectively, during the period of suckling and 15 days after weaning. Then the newborn ice were weighed at a litter. The blood sample was taken from the eye ball of the newborn mice. The increase of the body weight, and classification of white cells of newborn mice were shown as follows:
Results and discussion l. Results:
l. 1 Table 1 showed body weight changes of newborn mice.
Table l Effect of 851NS on body weight increase of the newborn mice brood numbernewborn mice body weight p value*
number increase of each newborn mouse (g. X+ SD) control 4 34 14.20+0.75 851 4 34 14.36+1.29 < 0.05 * Compared with control group 1.2 Table 2 showed Effect of 851NS on white cell classification of newborn mice Table 2 (% X + D.SD) group micemorphonaclear p 1 valuelymphocyte p 2 value*
numberleukocyte (% X +SD) (% X _SD) control 34 58+10 42+9 851 34 42+12 <0.001 57+12 <0.001 * Compared with control group 2. Discussion 851 nutrient solution was a bioengineering product which contained more than 20 kinds of amino acids, several kinds of vitamins and trace elements. The results showed that 851 nutrient solution significantly increased the value of lymphocoyte of the newborn mice, which indicated that 851 NS could stimulate and improve the blood manufacturing function of the newborn mice.
The results also showed that 851NS could increase the body weight of the newbornmice, which indicated that some components of 851NS were nutritious for the newborn mice.
~xperiment 6 Therapeutic effect of 851NS on the subchronic injury to gastric mucosa in mice 1. Materials Kunming mice of both sex weighing 42-44 g were supplied by the animal center of Fujian Medical College. 851NS, 4N HCI and 0.1% indomethacin were used.
2. Methods 2.1 The establishment of the model of subchronic injury to gastric mucasain mice Eight mice were divided into 2 groups in random. Mice of the control group were administered with normal saline once daily, P.O. Mice of the tested group were administered with 4N HCl or 0.1% indomethacin alternately once daily, (P.O.) for two days. These mice were fed with normal diet and tap water. Ten days later, the mice were decapitated. The stomach was removed and washed with water. The gastric mucosa of the control group showed lustrous and red, while the gastric mucosa of the tested group were lustreless, pale and swelling. The gastric cavity was fill with 10%
neutral formalin for fixing, cut into sections with paraffin wax and stained with HE
staining. Gastric mucosa were examined with microscope and showed that the endothilium of the mucosa became thinner and covered with eroded keratinic substances, gastric gland scattered with eroded necrosis. No significant changes was observed in control group. It was confirmed that the method described above could become a model of subchronic injury.
2.2 Therapeutic effect of 851NS on mice subchronic injury of gastric moucosa Thirty mice were divided into control group and 851 group in random and administered with HCl or indomethacin alternative according to the method described above for 10 days. The mice of these two groups were fed with normal diet or the diet containing 851, respectively during 10 days and continued for 7 days after the ~11mini~tration of HCl or indomethacin was stopped. Then the mice wére decapitated. The change of the gastric moucosa was examined according to the method described above.
Result and Discussion There was no significant difference of the morphology of gastric moucosa between the control and 851 groups. The mucosa in the front part of the stomach of the 851 group was a little thicker than that of the control group. The results indicated that 851 was beneficial to the recovery of injury of gastric mucosa.
Experiment 7 Therapeutic effect of 851 on hemmorhage of gastric mucosa in rats Materials and Methods 1. Methods Rats were divided into control and 851 groups in random and fed with normal diet and the diet containing 851 (8 ml/rat), respectively. All rats were supplied with sufficient tap water. 15 days later, all rats were fasted for 14 hrs. and administed with indomethacin (2.5 mg/rat) subcutaneously once daily for two days. 18 hours after the second injection, rats were decapitated and the stomach was examined.
Results and Discussion 1 . Results Number and degree of acute hemorrhage of gastric mucosa: The hemorrhage of gastric mucosa in control 6 mice and 4 mice in 851 group was locally restricted as spotted, stripped or stretched situation. Diffused hemorrhage of gastric mucosa occurred in one rat of the control group. There were 6-12 spots hemorrhage of gastric mucosa in control group and 2-8 in 851 group. The number of hemorrhage of gastric mucosa of control group was significantly severe than that of 851 group.
2. Discussion The results indicated that indomethasin induced hemorrhage of gastric mucosa wasprevented when rats were fed with 851NS for 15 days.
Experiment 8 Effect of 851 NS on CCl4 induced subchronic hepatic injury in rat Material and Methods 1. Materials Twenty two Wistar rats (male 10 and female 12), weighing 140-220 g were supplied by the animal center of Fujian Medical College. CCl4 analytical grade, was diluted in purified peanut oil to the concentration of 40%. 851 NS, agar gel (product of Sh~ngh~i Chemical Agent Manufactory) and LEB multiple usage of electrophoresis equipment were used.
2. Methods Twenty two rats were divided randomly into 3 groups, i.e. control group (n=6), CCl4 group (n=8) and CCl4 + 851 group (n=8). Rats of CCl4 and CCl4 + 851 group were administered with CCl4 (0.1 ml/lOOg) subcutaneously twice a week (3 days interval), for 5 weeks. Rats of control and CCl4 groups were fed with normal diet. Rats of CCl4 +
851 group were fed with the diet containing 851 NS (8 ml/rat.day). All rats weresupplied with sufficient tap water and fed for 5 weeks. Five days after the lastadministration of CC14, blood was taken from the axillary vein. Serum was collected and liver function was detected. Electrophoresis of serum protein was performed with LEB electrophoresis equipment. Pathological changes of the liver were examined.
Results and Discussion 1. Results:
l . l Rat SGPT
Effect of 851 NS on CCl4 induced SGPT changes in rats group number SGPT(%,X + SD) p l* p 2*
control 6 29.8 + 8.0 CCl4 8 41.6 + 5.3 < 0.01 CCl4 + 851 8 31.0 + 5.5 > 0.05 <0.01 * pl compared with control group, p2 compared with CCl4 group.
1.2 There were no significant differences of thymol turbidity test (l-l l) and zinc sulfate turbidity test (Zn TT) between these groups.
1.3 Observation of liver by naked eye: The color and lustre of the liver capsule in control group were normal. The liver capsule was lustrous. The rat liver capsules of CCl4 group were pale brown and lustrous in accordance with the morphological changes of liver with watery degeneration. The rat liver mophology of CCl4 + 851 group was better than that of CCl4 group and poor than that of control group.
1.4 Observation of liver by microscope: In control group the liver histological structure of 5 rats were normal except one with small watery degeneration. Eight rats of CCl4 group showed significant liver watery degeneration. Cell plasma of the outer part of liver lobule showed diffused or focal reticulated areosis. Some cells in the central part of the liver loblue were normal and some showed degeneration. Fatty degenerated liver cells were scattered or focused and difficult to be differentiated from watery degenerated cells. Six rat liver of the CCl4 + 851 group showed less watery degeneration than that of CCl4 group, another 2 rats showed the same watery degeneration as that of CCl4 group. Rat liver histological changes were in accordance with the results of liver function SGPT. This indicated that the results of rat SGPT in all groups were reliable.
1.5 Electrophoresis of rat serum protein: Two serum samples of each group were used for electrophoresis. The results showed that there were no differences among these groups. These results indicated that CCl4 induced liver injury did still not cause changes of rat serum protein.
1.6 Increase of body weight: The increase of rat body weight of CCl4 + 851 groupwere more than that of the other groups, but there were not statistically significant differences among these groups (p~0.05).
2. Discussion The results of rat serum SGPT and morphological changes investigated by naked eye and microscope indicated that 851 showed preventive and therapeutic effects against CCl4 induced subchronic liver injury.
~xperiment 9 Effect of 851 NS on the body weight development of newborn and suckling mice Materials and Methods l. Materials Kunming mice were supplied by the animal center of Fujian Medial College. 851 NS(batch number 06) was used.
2. Methods 27 female Kunming mice after mating with the same species of male mice, when they were preliminary considered to be pregnant (with vaginal thrombus), were divided into control group and 851 groups randomly. Mice of control group were fed with normal diet while mice of the 851 group were fed with the diet containing 851. All mice were supplied with tap water. Seven mice were taken from each group in random and fedwith the same method described above separately after they were confirmed to be pregnant from the enlarging abdomen. Newborn mice per litter were weighed at the day of parturition, and after the period of 20 days suckling. The body weight and the external appearance of the newborn mice and suckling mice were investigated.
Results and Discussion 1. Results 1.1Effect of 851NS on the body weight of the newborn mice (just after parturition) Table 3Effect of 851 on the body weight of newborn mice group litter number number of body weight of average body p*
newborn mice each newborn weight of each per litter mice in the newborn mice same litter (g.X) in the group (g.x_50) Control l 13 1.54 2 lO 1.70 3 9 1.70 1.61 + 0.067 4 10 1.60 1.65 6 10 1.57 7 10 1.54 851 l 12 1.66 2 ll 1.77 3 8 2.15 4 9 1.85 1.83+0.15 <0.01 8 1.81 6 10 1.78 7 13 1.78 * Compared with control group.
1.2 Effect of 851NS on the body weight of suckling mice were shown in table 4.
Table 4 Effect of 851NS on the body weight of suckling mice group litter number of body weight of average of body weight p*
number suckling the suckling mice of the suckling mice in mice in the same litter the same group (g.X+50) (g.X) control 1 10 10.60 2 9 11.30 3 11 8.45 4 8 12.60 10.52+ 1.68 8 11.20 6 13 8.00 7 8 11.50 851 1 9 13.00 2 9 12.85 3 9 11.53 4 8 14.75 12.44 + 1.81 <0.05 8 14.36 6 10 10.64 7 13 9.98 * Compared with control group.
1.3 No abnormal phenomenon of external appearance, capacity for eating and general activities were observed.
2. Discussion 2.1 Table 3 showed that 851NS could promote the body weight development of the mouse embryo when the female mice were fed with 851 NS during the period of pregnancy (p < 0.01).
2.2 Table 4 showed that 851 NS could promote the body weight development of the suckling mice when the female mice were fed with 851 NS during the period of thesuckling (p < 0.05).
2.3 The results indicated that no abnormal development (such as fetus malformation) and adverse effect were observed when female mice were fed with 851 NS during 40 days period from pregnancy to suckling.
The embodiments of the present invention are described, but not limited, in detail as follows:
Example 1 A 2.5 ton tank fermenter was used in this production. The seed fermenter was of 0.5 ton. The amount of raw materials taken in was 40%, a 5% soybean medium was adopted as the producing medium containing: 5% of soybean powder, 0.04% of yeastextract, 0.05 % KH2PO4, 0.02 % of MgSO4, 0.02 % of CaC03, 0.02 % of NaCl, 10 ppmof Na2MoO4, 2.5 ppm of Na2SeO3, 10 ppm of ZnSO4, 2.5 ppm of CoCl2 and water.
The amount of inoculum was 1 %, the seed fermenter was of 2 liters, the cell age was 24 hrs., ( the first seed culture -- the second seed culture -- the third seed culture, the culture incubated for 24 hrs. was used as inoculum.) culture te",pel~ture was 30~C, stirring speed was 260 rpm, the aeration rate was l: 0.8 wm. The culture incubation lasted for 45 hrs. After terminating the incubation, the culture was autoclaved and packed into bottles through an assembly line, then autoclaved again to give final products. 100 ml of the culture solution contained: 3.7% of dry material, 2.06% of proteins, 0.23% of lipids, 0.75% carbohydrates, vitamins and mineral salts. (listed in Table 5) Table 5 Analytical report of 851 culture solution Constituents Unit Amount ConstituentsUnit Amount eatable part g 100 asparic acid mg 87.8 water g 96.3 gulutamic acidmg 93.8 protein g 2.06 serine mg 28.1 lipid g 0.23 proline mg 29.0 carbohydrate g 0.75 glycine mg 33.6 heat of capacitykcal 13.3 alanine mg 48.1 eatable threonine mg 30.0 cellulose g 0.26 valine mg 37.0 ash g 0.40 methionine mg 10.9 isoleucine mg 26.1 K mg 49.0 leucine mg 51.4 Na mg 18.0 tyrosine mg 22.0 Ca mg 39.5 phenylalaninemg 24.0 Fe mg 2.3 lysine mg 34.7 Zn mg 4.4 tryptophen mg 10.0 Cu mg 0.05 histidine mg 14.2 Mn mg 0.14 arginine mg 39.6 Mg mg 5.3 P mg 42.9 myristic acid %
Se mg - 0.05 palmitic acid % 14.4 stearic acid % 85.5 vitamin E mg 0.64 vitamin B2 mg 0.13 Vitamin PP mg 0.47 Example 2 An 8 ton tank fermenter was used in this production. The amount taken in of raw materials was 40%. It means that the amount of medium was 3.2 ton. A 10% soybeanmilk medium was adopted. The medium contained: 320 kg of soybeans, 1.28 kg of yeast extract, 1.6 kg of KH2PO4, 640 g of MgSO4, 640 g of CaC03, 640 g of NaCl, 32 g of Na2MoO4, 8 g of Na2SeO3, 32 g of ZnSO4, 8 g of CoCl2 and water. The amount of inoculum was 1 %, the cell age was 24 hrs., (the first seed culture, the second seed culture and the third seed culture, the culture incubated for 24 hrs. was used as inoculum.) The culture temperature: 30~C, the stirring speed: 220 rpm, the aeration rate: 1:0.8 wm. After fini.~hing the incubation in a seed fermenter, the inoculum was inoculated into an 8 ton tank fermenter, the culture temperature: 30~C, the stirring speed: 220 rpm, the aeration rate: 1:0.8 vvm. The incubation was terminated after 48 hrs. and the culture solution was autoclaved and packed into bottles through an assembly line, then autoclaved again to give the final products. 100 ml of the culture solution contained: aspartic acid 543 mg, glutamic acid 710 mg, serine 160 mg, glycine 280 mg, histidine 65 mg, argining 226 mg, threonine 232 mg, alanine 359 mg, proline 126 mg, tyrosine 101 mg, valine 283 mg, methionine 53 mg, isoleucine 262 mg, leucine 309 mg, phenylalanine 148 mg, lysine 212 mg, cystine 38 mg, the total amount: 4127 mg.
Example 3 The nutrient solution produced by the method given in example 2 was dried by a spray dryer usually used to make dried milk to give 851 nutrient powder. The temperature was of 80~C at the spray dryer entrance and 60~C at the exit. 0.8 ton of 851 nutrient solution gave 20.4 kg of dried powder, which was further processed into capsules. One capsule contained 0.28 g of dried powder or formulated with 80 kg of starch and 10 kg of honey to make granule preparations.
Example 4 The 851 nutrient solution produced by conventional fermentation method was centrifuged to get rid of precipitate, at 15000 rpm. Thus 1 ton of 851 nutrient solution gave 960 kg of supernatant. 851 fruit juice cola was prepared by adding 500 kg of fruit juice, 851 honey cola was prepared by adding 40 kg of honey. The supernatant mentioned above might also be flavoured to give a variety of 851 drinks with different taste according to needs.
Example S
The 851 nutrient solution produced by conventional fermentation method was centrifuged. The supernatant was concentrated by a film evaporator or a vaccum rotatory evaporator to five 851 concentrated nutrients. After evaporation, 0.5 ton of the supernatant gave 100 kg of concentrated nutrient. After adding 10 kg of honey, the concentrated nutrient was packed into small bottles with easy-to-open cap. This nutrient is convenient to be taken orally.
Claims (18)
1. A strain 851R, a mutant of Microbacterium, having the capability to convert plant protein to single cell protein and possessing the following characteristics: a Gram-positive bacterium, motionless, able to produce H2S, nitrate reduction positive, alive in milk being heated to 60°C for 30 minutes or to 72°C for 15 minutes, the thallus being of red or orange red colour, the colour of the thallus being turned to pink at 38°C and to colourless at 40°C, the DNA of the strain containing R fragment.
2. The strain 851R, a mutant of Microbacterium in accordance with claim 1, wherein said R fragment in the DNA of the strain can be detected by a special R
fragment DNA probe.
fragment DNA probe.
3. The strain 851R in accordance with claim 1, wherein said plant protein is soybean protein.
4. A strain 851R, a mutant of Microbacterium, having the essential characteristics of the sample on deposit with American Type Culture Collection 12301 Parklawn Drive, Rockville, Maryland 20852 USA, and assigned A.T.C.C. No.53981.
5. A method for producing single cell protein nutrient solution by using bacterium, wherein the strain 851R, a mutant of Microbacterium, is adopted as a producing strain and a soybean powder medium or starch medium is adopted as producing media to produce 851R nutrient solution by fermentation.
6. The method in accordance with claim 5 wherein an oven dry spray processing to harvest single cell protein from culture solution and concentration processing to refine culture solution are adopted.
7. A medium formula for the strain 851R in accordance with claim 1, having the following components by weight:
Soybean powder 5 - 10%
or soybean milk (calculated as the weight of soybean) 5 - 20%
or various bean cakes sunflower seed cake 5 - 10%
Yeast extract or yeast powder 0.02 - 0.5%
CaCO3 0.05 - 0.5%
K2HPO4 0.02 - 0.2 %
MgSO4 0.01 - 0.08 %
NaCl 0.01 - 0.08 %
Na2MoO4 5 - 50 ppm Na2SeO3 1.5 - 10 ppm ZnSO4 2.5 - 50 ppm CoCl2 2.5 - 50 ppm
Soybean powder 5 - 10%
or soybean milk (calculated as the weight of soybean) 5 - 20%
or various bean cakes sunflower seed cake 5 - 10%
Yeast extract or yeast powder 0.02 - 0.5%
CaCO3 0.05 - 0.5%
K2HPO4 0.02 - 0.2 %
MgSO4 0.01 - 0.08 %
NaCl 0.01 - 0.08 %
Na2MoO4 5 - 50 ppm Na2SeO3 1.5 - 10 ppm ZnSO4 2.5 - 50 ppm CoCl2 2.5 - 50 ppm
8. A medium formula for the strain 851R in accordance with claim 1, having the following components percentage by weight:
Fresh materials containing starch 10 - 20%
(Sweet potato, potato, etc.) or dry materials containing starch 1- 10 %
(corn powder, etc.) Yeast extract 0.04 - 0.08%
CaCO3 0.05 - 0.5 %
K2HPO4 0.05 - 0.1 %
MgSO4 0.02 - 0.04%
NaCl 0.02 - 0.04%
Na2MoO4 10 - 20 ppm Na2SeO3 1.5 - 20 ppm ZnSO4 5 - 20 ppm CoCl2 5 - 20 ppm
Fresh materials containing starch 10 - 20%
(Sweet potato, potato, etc.) or dry materials containing starch 1- 10 %
(corn powder, etc.) Yeast extract 0.04 - 0.08%
CaCO3 0.05 - 0.5 %
K2HPO4 0.05 - 0.1 %
MgSO4 0.02 - 0.04%
NaCl 0.02 - 0.04%
Na2MoO4 10 - 20 ppm Na2SeO3 1.5 - 20 ppm ZnSO4 5 - 20 ppm CoCl2 5 - 20 ppm
9. A nutrient solution produced by fermentation with the strain 851R, a mutant of Microbacterium, in media containing nitrogen source.
10. The 851R nutrient solution in accordance with claim 9, wherein the solution has an anti-aging effect.
11. The 851R nutrient solution in accordance with claim 9, wherein the solution has an effect of improving constitutions.
12. The 851R nutrient solution in accordance with claim 9, wherein the solution has an effect of inhibiting cancer cell growth.
13. The 851R nutrient solution in accordance with claim 9, wherein the solution has an effect of stimulating and improving homogenises of organisms.
14. The 851R nutrient solution in accordance with claim 9, wherein the solution has an effect of restoration of gastric mucosa injury.
15. The 851R nutrient solution in accordance with claim 9, wherein the solution has a therapeutic action on acute bleeding of gastric mucosa.
16. The 851R nutrient solution in accordance with claim 9, wherein the solution has a therapeutic action on liver injury.
17. The 851R nutrient solution in accordance with claim 9, wherein the solution has an effect of restoration of puerperal constitution.
18. A series of products made of the bacteria of the strain 851R or 851 nutrient solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN89104615 | 1989-07-11 | ||
| CN89104615A CN1023409C (en) | 1989-07-11 | 1989-07-11 | Nutritive liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2012278A1 CA2012278A1 (en) | 1991-01-11 |
| CA2012278C true CA2012278C (en) | 1998-06-02 |
Family
ID=4855628
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002012278A Expired - Fee Related CA2012278C (en) | 1989-07-11 | 1990-03-15 | Mutant of microbacterium, a strain 851r, and a process for producing 851r nutrient solution by application of the strain |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JP2613664B2 (en) |
| CN (1) | CN1023409C (en) |
| AU (1) | AU624690B2 (en) |
| CA (1) | CA2012278C (en) |
| GB (1) | GB2233666B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6479050B1 (en) | 1991-06-03 | 2002-11-12 | Innovi N.V. | Trace element-rich additive, method for preparing same, preparation in which the additive is included and use thereof |
| CN1101471C (en) * | 2000-05-26 | 2003-02-12 | 黑龙江省轻工科学研究院 | Process for preparing active peptide of soybean protein |
| US7472891B2 (en) | 2006-01-30 | 2009-01-06 | Michael D. Schram | Bollard assembly |
| CN102719369B (en) * | 2011-06-14 | 2013-12-04 | 广西中烟工业有限责任公司 | Microbial strain and application thereof |
| US20140302171A1 (en) * | 2011-10-12 | 2014-10-09 | For Days Co., Ltd | Cysteine Peptide-Containing Health Drink |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4920052A (en) * | 1988-02-05 | 1990-04-24 | Miles Inc. | Process for producing glucose isomerase |
-
1989
- 1989-07-11 CN CN89104615A patent/CN1023409C/en not_active Expired - Fee Related
-
1990
- 1990-03-15 CA CA002012278A patent/CA2012278C/en not_active Expired - Fee Related
- 1990-03-22 AU AU52100/90A patent/AU624690B2/en not_active Ceased
- 1990-05-15 GB GB9010885A patent/GB2233666B/en not_active Expired - Fee Related
- 1990-07-11 JP JP2183854A patent/JP2613664B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CA2012278A1 (en) | 1991-01-11 |
| GB2233666B (en) | 1993-08-04 |
| CN1023409C (en) | 1994-01-05 |
| AU624690B2 (en) | 1992-06-18 |
| CN1042376A (en) | 1990-05-23 |
| AU5210090A (en) | 1991-01-17 |
| JPH0349680A (en) | 1991-03-04 |
| GB9010885D0 (en) | 1990-07-04 |
| JP2613664B2 (en) | 1997-05-28 |
| GB2233666A (en) | 1991-01-16 |
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