CN106173201A - A kind of fermentation process improving Thallus Gracilariae quality - Google Patents
A kind of fermentation process improving Thallus Gracilariae quality Download PDFInfo
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Abstract
The present invention relates to technical field of biological fermentation, be specifically related to a kind of fermentation process improving Thallus Gracilariae quality.Said method comprising the steps of: Thallus Gracilariae is cleaned by S1 dries, and is crushed to little granule；S2 makes fermentation medium, and Thallus Gracilariae granule adds distilled water, regulates salinity with NaCl, and NaCl consumption is 1 4% (w/v), mix homogeneously, regulates pH value 7.2 7.6；S3 autoclaving, inoculates zymocyte liquid 1 15% (v/v) after cooling；S4 ferments, and arranges temperature 25 DEG C, fermentation tank rotating speed 150r/min, fermentation time 12 48 hours.Compared with prior art, the present invention uses Flavobacterium to ferment, it is possible to the most quickly increases the content of oligosaccharide in Thallus Gracilariae, improves its nutritive value as economic fish feedstuff, and this strain need not agar, having had only to NaCl, source is very simple, and this fermentation process temperature is relatively low, speed is fast, step is simple, and operability is relatively strong, easily amplifies and realizes factorial praluction.
Description
Technical field
The present invention relates to technical field of biological fermentation, be specifically related to a kind of fermentation process improving Thallus Gracilariae quality.
Background technology
Thallus Gracilariae is relatively conventional tangleweed, is extensively ground because it has Important Economic value and ecological functions
Study carefully, and realize artificial culture cultivation in China's Coastal Areas, become another the emerging sea after Thallus Laminariae (Thallus Eckloniae), Thallus Porphyrae, Thallus Laminariae
Algae industrial colony.Thallus Gracilariae main component is agar polysaccharide and crude fibre, accounts for the 59.4% of frond.The protein content of Thallus Gracilariae
For 14.5-22.8%, aminoacid composition equilibrium, fat content is relatively low, for 0.4-0.8%.Meanwhile, Thallus Gracilariae vitamin and mineral nitrogen
Constituent content enriches, particularly the element such as Mg, Fe, Ca, K, Na, Zn.Therefore, Thallus Gracilariae is a kind of high dietary-fiber, high protein
Matter, the low-fat seaweed fodder raw material rich in vitamin and mineral, make full use of these advantages, can it be opened further
Send out and utilize.But, agar polysaccharide too high in Thallus Gracilariae and crude fibre can reduce its digesting and assimilating in animal intestines and stomach, reduce
Its nutritive value.
Summary of the invention
It is an object of the invention to provide a kind of fermentation process improving Thallus Gracilariae quality, utilize the characteristic of Flavobacterium, low
Temperature fermentation, quickly reduces glue polysaccharide and crude fiber content in Thallus Gracilariae, improves the widow that in Thallus Gracilariae, animal can digest and absorb
Sugar content and protein content, decompose feedstock portions harmful substance, reduces its toxicity, improves the Thallus Gracilariae battalion as feedstuff
Support and be worth and value.
In order to realize above-mentioned purpose, adopt the following technical scheme that.A kind of fermentation process improving Thallus Gracilariae quality, institute
The method of stating comprises the following steps:
The preparation of 1 Thallus Gracilariae raw material
1. Thallus Gracilariae gathers: after Thallus Gracilariae gathers, and cleans, goes the removal of impurity, dry；
2. prepared by Thallus Gracilariae powder: the Thallus Gracilariae dried being put into drying baker and dries, moisture controls, 10～15%, then to use
Pulverizer is crushed to 40～100 mesh, standby.
The selection of 2 fermented bacteriums
The preferred strain of the present invention is ocean agarase Producing Strain Flavobacterium (Flavobacterium sp.), and this strain is by Shan
Biology department of college of science of head university is screened, and is deposited in China Committee for Culture Collection of Microorganisms on January 16th, 2008 general
Logical microorganism center, bacterial strain deposit number is: CGMCC NO.2342.Patent CN101608166A discloses the separation of this strain
Screening and authentication method.
2.1 strain activation and culture bases
Seed culture medium (2216E culture medium) forms: peptone 5g, yeast powder 1g, iron phosphate 0.01g, Chen Haishui 1000ml, adjusts
Joint pH is 7.2～7.6,121 DEG C of sterilizing 20min；
Shake flask medium: 2216E culture medium adds the agar powder of 0.2%；
Solid medium: 2216E culture medium adds the agar powder of 1.5%；
Fermentation medium: Thallus Gracilariae content 3%, NaCl content 2%, distilled water preparation, pH7.6,121 DEG C of sterilizing 20min are standby.
2.2 cultural method
S1 shake flask fermentation is cultivated: access in 2216E culture medium by-80 DEG C of antibacterials preserved by the inoculum concentration of 2%, 25 DEG C, constant temperature
In shaking table incubator, 150r/min cultivates 58 hours, under 600nm wavelength, by spectrophotometric determination OD value, within every 6 hours, measures
Once and record, measurement result is shown in Table 1, and bacterium solution is the highest at 40 hour concentrations as can be seen from Table 1；
S2 actication of culture: the antibacterial line preserved-80 DEG C is connected to the activation of solid medium flat board, transfers in 100ml the most again
Shake flask medium continues activation culture 40h in 25 DEG C, obtains bacterium solution；
S3 glycerol conservation: the glycerite of cultured bacterium solution and 50% is divided in seed bottle in the ratio mixing of 1:1, protects
Be stored in-80 DEG C standby.
The concentration change of the different incubation time bacterium solution of table 1
3 single factor experiment optimization of fermentation conditions
Thallus Gracilariae addition, strain inoculum concentration, NaCl addition, Thallus Gracilariae are pulverized 5 factors such as granular size, fermentation time
Carry out single factor experiment optimization successively, according to thick protein, oligosaccharide content and crude fibre in Thallus Gracilariae fermentation liquid after fermentation
Content, filters out optimal fermentation condition.Each time after fermentation ends, take the drying of 65 DEG C of Partial fermentation sample, pulverized 40 mesh
Sieve, is used for measuring thick protein, oligosaccharide and crude fibre content.
The optimization of 3.1 Thallus Gracilariae additions
The bacterium solution of activation will be cultivated, access in the Thallus Gracilariae culture medium containing 2% (w/v) NaCl by the inoculum concentration of 5% (v/v), if
Put the Thallus Gracilariae addition (w/v) of 5 different gradients, respectively 1%, 2%, 3%, 4%, 5%, each process 3 repetition.
PH7.6,25 DEG C, after cultivating 30 hours under 150r/min rotating speed in constant-temperature table incubator, measurement result is shown in Table 2.
Table 2 different Thallus Gracilariae addition tunning change
Note: with in column data, represent mutual significant difference (P < 0.05) without identical lower case mark person.
As shown in Table 2, crude protein content gradually rises along with the increase of Thallus Gracilariae addition, and when 3%, content is the highest, for
16.43%, reduce the most again；Oligosaccharide content constantly raises along with the increase of Thallus Gracilariae addition, and when 3%, content is the highest, for
2.07%, reduce the most again；Crude fibre content is gradually lowered along with the increase of Thallus Gracilariae addition, and when 3%, content is minimum,
It is 3.75%, raises the most again.Therefore, it is 3% preferable for selecting Thallus Gracilariae addition.
3.2 Thallus Gracilariaes pulverize granular size optimization
By cultivating the bacterium solution of activation, it is inoculated in containing 3% (w/v) Thallus Gracilariae by the inoculum concentration of 5% (v/v), 2% (w/v) NaCl's
In fermentation medium, the Thallus Gracilariae pulverized particles of 4 different proportions is set, respectively 40,60,80,100 mesh, each process 3
Repeat.PH7.6,25 DEG C, after cultivating 30 hours under 150r/min rotating speed in constant-temperature table incubator, measurement result is shown in Table 3.
The tunning change of the different Thallus Gracilariae granular size of table 3
Note: with in column data, represent mutual significant difference (P < 0.05) without identical lower case mark person.
As shown in Table 3, crude protein and oligosaccharide content are the highest when Thallus Gracilariae pulverized particles crosses 40 mesh, are 16.38% He
1.88%, along with Thallus Gracilariae pulverized particles is the thinnest, crude protein and oligosaccharide content all reduce；Crude fibre content is pulverized Thallus Gracilariae
Granule is crossed during 40 mesh minimum, is 3.88%, and along with Thallus Gracilariae pulverized particles is the thinnest, crude fibre content raises.Therefore, Thallus Gracilariae
It is preferable that pulverized particles crosses 40 mesh.
The optimization of 3.3 fermentation medium NaCl additions
This method uses adds the NaCl method of different content to substitute Chen Haishui in distilled water, thus Optimal Medium.If
Put the NaCl addition (w/v) of 4 different proportions, respectively 1%, 2%, 3%, 4%, by cultivating the bacterium solution of activation, by 5%
(v/v) inoculum concentration is transferred in containing in 3% (w/v) Thallus Gracilariae fermentation medium, each process 3 repetition.PH7.6,25 DEG C, permanent
After cultivating 30 hours under 150r/min rotating speed in temperature shaking table incubator, measurement result is shown in Table 4.
Table 4 adds the tunning change of different NaCl content
Note: with in column data, represent mutual significant difference (P < 0.05) without identical lower case mark person.
As shown in Table 4, crude protein content gradually rises along with the increase of fermentation medium NaCl addition, contains when 3%
Measure the highest, be 16.50%, reduce the most again；Oligosaccharide content constantly raises along with the increase of fermentation medium NaCl addition,
When 3%, content is the highest, is 1.97%, reduces the most again；Crude fibre content is along with the increase of fermentation medium NaCl addition
And be gradually lowered, when 3%, content is minimum, is 3.77%, raises the most again.Therefore, select fermentation medium adds 3%
NaCl is preferable.
The optimization of 3.4 zymocyte liquid inoculum concentrations
The bacterium solution of activation will be cultivated, the bacterium solution inoculum concentration (v/v) of 5 different proportions will be set, respectively 1%, 2%, 5%, 10%,
15%, it is inoculated in respectively containing 3% (w/v) Thallus Gracilariae, in the fermentation medium of 3% (w/v) NaCl, each process 3 repetition.
PH7.6,25 DEG C, after cultivating 30 hours under 150r/min rotating speed in constant-temperature table incubator, measurement result is shown in Table 5.
The tunning change of different content bacterium solution inoculated by table 5
Note: in colleague's data, represent mutual significant difference (P < 0.05) without identical lower case mark person.
As shown in Table 5, crude protein content gradually rises along with the increase of zymocyte liquid inoculum concentration, and when 10%, content is
Height, is 16.86%, reduces the most again；Oligosaccharide content constantly raises along with the increase of zymogenous bacteria inoculum concentration, and when 10%, content is
Height, is 2.13%, reduces the most again；Crude fibre content is gradually lowered, when 10% along with the increase of zymogenous bacteria inoculum concentration
Content is minimum, is 3.88%, raises the most again.Therefore, zymogenous bacteria inoculum concentration selects preferable 10%.
3.5 the optimization of fermentation time
To cultivate the bacterium solution of activation, access containing 3% (w/v) Thallus Gracilariae by the inoculum concentration of 10% (v/v), 3% (w/v) NaCl sends out
In ferment culture medium, 9 fermentation time points, respectively 12h, 24h, 36h, 48h, 60h, 72h, 84h, 3 weights of each process are set
Multiple.PH7.6, cultivates under 150r/min rotating speed in constant-temperature table incubator by 25 DEG C, every a period of time sampling detection, measurement result
It is shown in Table 6.
The product change of table 6 different fermentations time
Note: with in column data, represent mutual significant difference (P < 0.05) without identical lower case mark person
As shown in Table 6, crude protein content gradually rises along with the prolongation of fermentation time, and 72h content is the highest, for
16.50%, reduce the most again；Oligosaccharide content constantly raises along with the prolongation of fermentation time, and 72h content is the highest, is 2.97%, with
After reduce again；Crude fibre content is gradually lowered along with the prolongation of fermentation time, and 72h content is minimum, is 3.81%, the most again
Raise.Therefore, fermentation time selects at 72 hours preferably.
4 optimization of orthogonal test fermentation conditions
On the basis of single factor experiment, select Thallus Gracilariae addition (A), bacterium solution inoculum concentration (B), NaCl concentration (C), Thallus Gracilariae
Four factors of granular size (D) carry out the orthogonal design of 4 factor 3 levels, further optimization of fermentation conditions, often 3 repetitions of group.Just
Experimental factor water-glass is handed over to be shown in Table 7.Fermentation temperature 25 DEG C, pH7.6, constant-temperature table incubator rotating speed 150r/min ferment 72 little
Time.After fermentation ends, take the drying of 65 DEG C of the fresh sample of part, be used for measuring oligosaccharide content.
The factor of table 7 Orthogonal Experiment and Design and level
Table 8 orthogonal test yields of oligosaccharides range analysis table
Table 9 yields of oligosaccharides analysis of variance table
From range analysis (table 8) oligosaccharide content R value, the sequencing affecting result of the test factor is: A > C > B > D,
The factor i.e. affecting Thallus Gracilariae fermentation product oligosaccharide is followed successively by: Thallus Gracilariae addition, NaCl add concentration, bacterium solution inoculum concentration, dragon palpus
Chinensis particle size.
It is A3B3C2D1 that K value according to oligosaccharide content can obtain best of breed, i.e. fermentation Thallus Gracilariae content 3%, NaCl concentration
2%, bacterium solution inoculum concentration 10%, Thallus Gracilariae granular size 40 mesh.Fermentation temperature 25 DEG C, pH7.6, constant-temperature table incubator rotating speed
150r/min, fermentation time 72 hours.After Thallus Gracilariae fermentation condition optimization, oligosaccharide content reaches 2.42%, and optimizes front oligosaccharide
Content is 0.81%, and oligosaccharide content improves 1.99 times.
By the F value of variance analysis (table 9) it can be seen that the sequencing affecting result of the test factor is: A > C > B > D, i.e.
The factor affecting Thallus Gracilariae fermentation product oligosaccharide is followed successively by: Thallus Gracilariae addition, NaCl concentration, bacterium solution inoculum concentration, Thallus Gracilariae granule
Size, and four factors are the most notable on the impact of Thallus Gracilariae fermentation product oligosaccharide.
Obtain the optimum condition of Thallus Gracilariae fermentation in conjunction with single factor experiment, ferment Thallus Gracilariae content 3%, NaCl concentration 2%,
Fermented bacterium inoculum concentration 10%, Thallus Gracilariae granular size 40 mesh.Fermentation temperature 25 DEG C, fermentation time 72 hours, fermentation liquid
PH7.6, fermentation tank rotating speed 150r/min.
5 determine optimum fermentation condition
The optimal conditions that Thallus Gracilariae is fermented is determined: ferment Thallus Gracilariae content in conjunction with single factor design and Orthogonal Experiment and Design
3%, NaCl concentration 3%, fermented bacterium inoculum concentration 10%, Thallus Gracilariae granular size 40 mesh, fermentation temperature 25 DEG C, fermentation time 72
Hour, fermentation liquid pH7.6, constant-temperature table incubator 150r/min.With this condition, 5L fermentation cylinder for fermentation Thallus Gracilariae, if 3
Repeat.After fermentation ends, tunning 65 DEG C drying, pulverize 40 mesh sieves, be used for measuring dry, coarse ash, crude fibre, thick
Fat, crude protein, oligosaccharide content, measurement result is shown in Table 10.
Table 10 feedstuff fermentation Thallus Gracilariae, the nutritional labeling of Thallus Gracilariae
As shown in Table 10, with optimal conditions ferment after, compared with matched group, on a dry matter basis, coarse ash by
30.4% brings up to 34.3%, and thick protein is brought up to 16.42% by 15.22%, and crude fat is reduced to 0.47% by 0.57%,
Oligosaccharide is brought up to 1.90% by 0.83%, and crude fibre is reduced to 3.87% by 6.32%.
In sum, the present invention utilizes ocean agarase Producing Strain Flavobacterium (Flavobacterium sp.) to ferment
Technology improves Thallus Gracilariae quality, and fermentation process comprises the following steps:
Thallus Gracilariae is cleaned by S1 dries, and is crushed to little granule；
S2 makes fermentation medium, and Thallus Gracilariae granule adds distilled water, regulates salinity with NaCl, and NaCl consumption is 1-4% (w/
V), mix homogeneously, regulate pH value 7.2-7.6；
S3 autoclaving, inoculates zymocyte liquid 1-15% (v/v) after cooling；
S4 ferments, and arranges temperature 25 DEG C, fermentation tank rotating speed 150r/min, fermentation time 12-48 hour.
Wherein, the zymocyte liquid in S3 is prepared by following methods: Flavobacterium is inoculated in 2216E solid medium flat board and lives
Change, transfer the most again in 100ml Shake flask medium, pH7.6, lives under 150r/min rotating speed in constant-temperature table incubator by 25 DEG C
Change and cultivate 40 hours, obtain bacterium solution.
As preferably, in step S1, after described Thallus Gracilariae is dried, moisture controls 10～15%, and pulverized particles is big
Little is 40 mesh.
As preferably, in step S1, in described fermentation medium, the addition of Thallus Gracilariae is 3% (w/v).
As preferably, in step S2, in described fermentation medium, NaCl addition is 2% (w/v).
As preferably, in step S2, described fermentation medium original ph is 7.6.
The Thallus Gracilariae tunning prepared according to above-mentioned fermentation process, than the nutritional labeling in unleavened Thallus Gracilariae more
Ideal, is more suitable for the culturing feed of economic fish, and by fermentation, in Thallus Gracilariae, agar polysaccharide and crude fiber content reduce
, increase oligosaccharide and protein content that animal can digest and absorb, improve albumen in tunning the most afterwards
Matter content, can decompose part harmful substance in raw material by fermentable in addition, reduce its toxicity.
Compared with prior art, the present invention uses single culture fermentation process Thallus Gracilariae, and the Flavobacterium fermentation of selection is permissible
Produce agarase, it is possible to the most quickly increase the content of oligosaccharide in Thallus Gracilariae, improve its nutriture value as economic fish feedstuff
Value, and this strain need not agar, it is only necessary to and having NaCl, source is very simple, and Thallus Gracilariae tunning can as feedstuff
Improving nutritive value not pollute the environment again, this fermentation process temperature is relatively low, and speed is fast, and step is simple, and operability is relatively strong, holds
Easily amplify and realize factorial praluction, Thallus Gracilariae is had important meaning as the further development and application of animal feed.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the invention will be further described.
This enforcement is with preferred parameter as fermentation condition, and concrete fermentation process includes following step:
Thallus Gracilariae is cleaned and dries by S1, puts into drying baker and dries to moisture 10-15%, then with pulverizer to granular size
40 mesh；
S2 makes fermentation medium, adds distilled water according to Thallus Gracilariae 3% (w/v), regulates salinity with NaCl, and NaCl consumption is
2% (w/v), mix homogeneously, regulate pH value 7.6；
Flavobacterium is inoculated in the activation of 2216E solid medium flat board by S3, transfers the most again in 100ml Shake flask medium,
PH7.6,25 DEG C, in constant-temperature table incubator, activation culture 40 hours under 150r/min rotating speed, obtain bacterium solution；
S4 autoclaving, 121 DEG C 20 minutes, after cooling inoculate zymocyte liquid 10% (v/v)；
S5 ferments, and arranges temperature 25 DEG C, fermentation tank rotating speed 150r/min, fermentation time 72 hours.
If 3 repetitions, 5L fermentation cylinder for fermentation Thallus Gracilariae, after fermentation ends, tunning 65 DEG C drying, pulverized 40
Mesh sieve, is used for measuring dry, coarse ash, crude fibre, crude fat, crude protein, oligosaccharide content, after fermenting with optimal conditions, with
Matched group is compared, and on a dry matter basis, coarse ash is brought up to 34.3% by 30.4%, and thick protein is brought up to by 15.22%
16.42%, crude fat is reduced to 0.47% by 0.57%, and oligosaccharide is brought up to 1.90% by 0.83%, and crude fibre is by 6.32% fall
Low to 3.87%.
Claims (9)
1. the fermentation process improving Thallus Gracilariae quality, it is characterised in that said method comprising the steps of:
Thallus Gracilariae is cleaned by S1 dries, and is crushed to little granule；
S2 makes fermentation medium, and Thallus Gracilariae granule adds distilled water, regulates salinity with NaCl, and NaCl consumption is 1-4% (w/
V), mix homogeneously, regulate pH value 7.2-7.6；
S3 autoclaving, inoculates zymocyte liquid 1-15% (v/v) after cooling；
S4 ferments, and arranges temperature 25 DEG C, fermentation tank rotating speed 150r/min, fermentation time 12-48 hour.
Fermentation process the most according to claim 1, it is characterised in that the strain of described zymocyte liquid is that ocean agarase is high
Producing bacterium Flavobacterium, it is the most micro-that this strain is deposited in China Committee for Culture Collection of Microorganisms on January 16th, 2008
Bio-Centers, bacterial strain deposit number is: CGMCC NO.2342.
Fermentation process the most according to claim 2, it is characterised in that in step S1, after described Thallus Gracilariae is dried, moisture contains
Amount controls 10～15%, and pulverized particles size is 40 mesh.
Fermentation process the most according to claim 2, it is characterised in that in step S1, Thallus Gracilariae in described fermentation medium
Addition be 3% (w/v).
Fermentation process the most according to claim 2, it is characterised in that in step S2, in described fermentation medium, NaCl adds
Dosage is 2% (w/v).
Fermentation process the most according to claim 2, it is characterised in that in step S2, described fermentation medium original ph
It is 7.6.
Fermentation process the most according to claim 2, it is characterised in that described zymocyte liquid is prepared by following methods: yellow bar
Bacterium is inoculated in the activation of 2216E solid medium flat board, transfers the most again in 100ml Shake flask medium, pH7.6,25 DEG C, constant temperature
In shaking table incubator, activation culture 40 hours under 150r/min rotating speed, obtain bacterium solution.
8. the Thallus Gracilariae tunning that the method as described in claim 1-7 any one prepares.
9. can be used for a feedstuff for economic fish cultivation, described feedstuff includes the Thallus Gracilariae tunning described in claim 8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159827A (en) * | 2020-10-16 | 2021-01-01 | 福建环海生物科技股份有限公司 | Production process of agar oligosaccharide |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608166A (en) * | 2008-05-15 | 2009-12-23 | 汕头大学 | One Flavobacterium strain and the application in producing agarase thereof |
| CN102827899A (en) * | 2012-09-18 | 2012-12-19 | 国家海洋局第三海洋研究所 | Gracilaria lemaneiformis agar oligosaccharide and preparation method and application thereof |
| CN103947873A (en) * | 2014-04-11 | 2014-07-30 | 汕头大学 | Gracilaria lemaneiformis-containing forage for lutianus stellatus Akazaki and preparation method thereof |
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2016
- 2016-07-04 CN CN201610518130.3A patent/CN106173201A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101608166A (en) * | 2008-05-15 | 2009-12-23 | 汕头大学 | One Flavobacterium strain and the application in producing agarase thereof |
| CN102827899A (en) * | 2012-09-18 | 2012-12-19 | 国家海洋局第三海洋研究所 | Gracilaria lemaneiformis agar oligosaccharide and preparation method and application thereof |
| CN103947873A (en) * | 2014-04-11 | 2014-07-30 | 汕头大学 | Gracilaria lemaneiformis-containing forage for lutianus stellatus Akazaki and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159827A (en) * | 2020-10-16 | 2021-01-01 | 福建环海生物科技股份有限公司 | Production process of agar oligosaccharide |
| CN112159827B (en) * | 2020-10-16 | 2022-03-08 | 福建环海生物科技股份有限公司 | Production process of agar oligosaccharide |
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Application publication date: 20161207 |