CN113045773B - Method for in-situ inducing nano-cellulose gelatinization in streptococcus zooepidemicus fermentation process - Google Patents

Method for in-situ inducing nano-cellulose gelatinization in streptococcus zooepidemicus fermentation process Download PDF

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CN113045773B
CN113045773B CN202110280726.5A CN202110280726A CN113045773B CN 113045773 B CN113045773 B CN 113045773B CN 202110280726 A CN202110280726 A CN 202110280726A CN 113045773 B CN113045773 B CN 113045773B
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streptococcus zooepidemicus
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CN113045773A (en
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李鑫
汤学宇
范一民
刘亮
王志国
勇强
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Nanjing Forestry University
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Abstract

The invention discloses a method for in-situ inducing nano-cellulose gelatinization in a Streptococcus zooepidemicus fermentation process, wherein nano-cellulose is added in the Streptococcus zooepidemicus (Streptococcus zooepidemicus) fermentation process. By the method, gel can be formed by adding the nano-cellulose, the yield of the hyaluronic acid is greatly improved, and the method has the characteristics of high product yield, mild conditions, strong operability, economy, environmental friendliness and the like.

Description

Method for in-situ inducing nano-cellulose gelatinization in streptococcus zooepidemicus fermentation process
Technical Field
The invention belongs to the technical field of biology and chemical industry, and particularly relates to a method for in-situ inducing nano-cellulose gelatinization in a streptococcus zooepidemicus fermentation process.
Background
Hyaluronic Acid (HA), also known as hyaluronic acid, is a linear macromolecular acidic mucopolysaccharide widely distributed in the human and animal body. In recent years, hyaluronic acid is widely used in the pharmaceutical and cosmetic industries because of its extremely strong water-retaining ability. The nanocellulose (TOCN) is used as a novel biomass nano material, and the nanocellulose not only has the characteristics of renewable nature cellulose, biodegradability and the like, but also has the advantages of large specific surface area, high hydrophilicity, high transparency, high strength, high young modulus, low thermal expansion coefficient and the like, provides possibility for forming various functional composite materials, and is proved to be self-assembled hydrogel or bio-based nano filler with wide prospect.
Hydrogels are a class of polymer materials having a three-dimensional network structure made of synthetic macromolecules and natural macromolecules, which can absorb a large amount of liquid and retain the liquid without being dissolved in a short time. Due to its high water content and high elastic behavior, hydrogels are widely used in tissue filling materials, drug carriers, cartilage regeneration, wound dressings, environmental adsorbents, and the like. The hyaluronic acid and the nano-cellulose can be combined to prepare a novel green, safe, healthy and nontoxic hydrogel composite material.
At present, starch-based hydrogel, cellulose-based hydrogel, etc. of polysaccharides are basically environmentally-friendly and degradable in starting preparation materials, and raw material sources are wide and renewable, but to prepare hydrogel with appropriate performance (such as appropriate pressure water retention, medium water absorption resistance, environmental stimulus responsiveness, etc.), chemical reactions such as corresponding modification and grafting (such as starch derivative hydrogel, modified cellulose-based hydrogel, etc.) are still required to be performed through subsequent chemical means, a large amount of chemical substances and specific environmental preparation conditions are inevitably used in the preparation process, and the greenness of the whole preparation process needs to be further improved.
In summary, in the preparation process of the existing polysaccharide hydrogel, a large amount of chemical substances and specific environments are inevitably used, so the invention provides a method for promoting the gelation of the nano cellulose while producing hyaluronic acid by streptococcus zooepidemicus fermentation, so as to effectively solve the problems and achieve the aims of high efficiency, strong operability, economy and environmental protection.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of the prior art and provides a method for inducing nano cellulose gelatinization in situ in the streptococcus zooepidemicus fermentation process.
The invention idea is as follows: the streptococcus zooepidemicus is used as a main body, fermentation is carried out in a culture medium to generate lactic acid and hyaluronic acid, the lactic acid and the hyaluronic acid cooperate with exogenously added nano-cellulose (the hyaluronic acid and the nano-cellulose can form composite dispersion liquid, and the lactic acid can provide hydrogen bonds for gel formation of the dispersion liquid), and the composite gel is formed under the action of the lactic acid.
In order to solve the technical problems, the invention discloses a method for inducing nano-cellulose gelatinization in situ in a Streptococcus zooepidemicus fermentation process, namely, nano-cellulose is additionally added in the process of producing hyaluronic acid and lactic acid by fermenting Streptococcus zooepidemicus (Streptococcus zooepidemicus).
Wherein, the Streptococcus zooepidemicus (Streptococcus zoepidemicus) includes but is not limited to ATCC 39920.
Wherein, seed liquid of Streptococcus zooepidemicus (Streptococcus zoepidemicus) is inoculated into a fermentation medium according to the volume ratio of 1-10%.
Preferably, seed liquid of Streptococcus zooepidemicus (Streptococcus zoepidemicus) is inoculated into the fermentation medium at a volume ratio of 5%.
Wherein the fermentation medium for fermentation comprises the following components in percentage by weight: 5-20g/L glucose, 5-20g/L peptone, 1-10g/L beef powder and 1-10g/L, NaH yeast powder2PO4 1-5g/L、NaCl 1-5g/L、MgSO4·7H2O 0.1-2g/L。
Preferably, the content of each component in the fermentation medium for fermentation is as follows: 10g/L glucose, 10g/L peptone, 5g/L beef powder and 5g/L, NaH yeast powder2PO4 2g/L、NaCl 2g/L、MgSO4·7H2O 1g/L
Wherein, in the whole fermentation process, the fermentation temperature is 30-40 ℃ no matter before or after the nano-cellulose is added.
Wherein, before adding the nano-cellulose, the rotation speed of the fermentation is 120-300 rpm.
Preferably, the rotation speed of the fermentation is 150rpm before adding the nanocellulose.
Wherein, the nano-cellulose is added in the middle and later period of the fermentation.
Preferably, the nanocellulose is added after fermentation for 10-30 h.
Further preferably, the nanocellulose is added after 24h of fermentation.
Wherein the nanocellulose is added in the form of a nanocellulose dispersion.
Wherein the solvent of the nano-cellulose dispersion liquid is distilled water.
Wherein the mass content of the nano-cellulose in the nano-cellulose dispersion liquid is 0.1-2%.
Preferably, the mass content of the nanocellulose in the nanocellulose dispersion liquid is 0.5% -2%.
Wherein the addition amount of the nano-cellulose is controlled to ensure that the final mass content of the nano-cellulose in the system is 0.1-1%.
Preferably, the addition amount of the nanocellulose is controlled so that the final mass content of the nanocellulose in the system is 0.5%.
Wherein, after the nano-cellulose is added, the rotation speed of the fermentation is 80-119 rpm.
Preferably, the rotation speed of the fermentation is 110rpm after adding the nanocellulose.
Wherein, after the nano-cellulose is added, the fermentation is continued for 2 to 20 hours.
Preferably, fermentation is continued for 10h after addition of nanocellulose.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the method for in-situ inducing gelation of the nano-cellulose by the microorganisms, the gel can be formed by adding the nano-cellulose, the yield of the hyaluronic acid is greatly improved, and the method has the characteristics of high product yield, mild conditions, strong operability, economy, environmental friendliness and the like.
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The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Fig. 1 is an SEM picture after induction of gelation by streptococcus zooepidemicus with the addition of 0.5% nanocellulose (a) and 2% nanocellulose (b).
FIG. 2 shows HA production with different amounts of nanocellulose.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The streptococcus zooepidemicus used in the examples described below is ATCC 39920.
Preparation of the media used in the following examples: 10g/L glucose, 10g/L peptone, 5g/L beef powder and 5g/L, NaH yeast powder2PO4 2g/L、NaCl 2g/L、MgSO4·7H2O1 g/L. The pH was adjusted to 7.0 with sodium hydroxide and sterilized.
In the following examples, the particle size of the nanofiber is 200-500 nm.
In the following examples, "%" refers to mass%.
In the following examples, the solvent of the nanocellulose dispersion was distilled water.
Example 1: fermenting and in-situ inducing nanometer cellulose gelatinization by using streptococcus zooepidemicus, comprising the following steps:
(1) inoculating the glycerol bacterium streptococcus zooepidemicus ATCC 39920 stored in a refrigerator at the temperature of-80 ℃ to a solid culture medium for culture to obtain a seed solution of the streptococcus zooepidemicus ATCC 39920;
(2) the resulting seed solution was inoculated into a shake flask containing 100mL of seed medium at an inoculum size of 5% v/v. Culturing on a shaking table under the following conditions: the rotating speed is 150rpm, the initial pH is 7.0, the fermentation temperature is 36 ℃, and the culture time is 24 hours;
(3) and (3) directly pouring the nano-cellulose dispersion liquid with the concentration of 0.5% into the shake flask in the step (2), enabling the final concentration of the nano-cellulose in the system to be 0.2%, reducing the rotating speed to be 100rpm, continuing to culture for 10h, finally obtaining a light yellow gelatinous substance, and simultaneously enabling the final yield of HA to be 3.26 g/L.
Example 2:
the shake flask fermentation process was identical to that of example 1, except that in step (3), the 2% nanocellulose dispersion was poured directly into the shake flask of step (2) to give a final nanocellulose concentration of 0.5% in the system, the rotation speed was reduced to 100rpm, and the cultivation was continued for 10 hours to give a nearly colorless transparent gel, while the final HA yield was 4.96 g/L.
Example 3
The gels of examples 1, 2 were characterized. Fig. 1 is SEM pictures of streptococcus zooepidemicus induced gelation after 0.5% nanocellulose (initial concentration) and 2% nanocellulose (initial concentration), and it can be clearly seen from a, b, two figures that at 0.5% nanocellulose addition, cellulose is wrapped by most HA, exhibiting a lamellar structure, whereas when the nanocellulose content is increased to 2%, the gel exhibits a dense network skeleton structure. Meanwhile, the streptococcus zooepidemicus fermentation can be used for regulating and controlling the in-situ induced gelation of the nanocellulose.
Example 4
The shake flask fermentation method was the same as that of example 1, except that in step (3), the 0.2% nanocellulose dispersion was directly poured into the shake flask of step (2) to make the final concentration of nanocellulose in the system 0.08%, and the cultivation was continued for 10h, and the final gel strength was too low to maintain its shape.
Comparative example 1
The shake flask fermentation process remained as in example 1 except that step (3) was carried out without adding nanocellulose, the rotation speed was directly reduced to 100rpm, and the cultivation was continued for 10 hours, and finally no clear gel could be obtained, while the final HA yield was 2.022 g/L.
When the hyaluronic acids of example 1, example 2 and comparative example 1 were measured, as shown in fig. 2, it was found that the yield of hyaluronic acid was increased by 52.14% compared to 0.5% when 2% of nanocellulose was added.
The invention provides a method and a thought for in-situ inducing nanometer cellulose gelatinization in a streptococcus zooepidemicus fermentation process, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that for a person skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the invention, and the improvements and modifications should be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims (4)

1. A method for in-situ inducing nano cellulose gelatinization in streptococcus zooepidemicus fermentation process is characterized in that streptococcus zooepidemicus (S.zooepidemicus)Streptococcus zooepidemicus) The seed liquid is inoculated into a fermentation culture medium for fermentation according to the volume ratio of 1-10 percent, and after the fermentation is carried out for 10-30h, nano-cellulose is added for continuous fermentation for 2-20 h;
wherein the content of the first and second substances,
controlling the addition amount of the nano-cellulose to ensure that the final mass content of the nano-cellulose in the system is 0.1-1%;
the fermentation temperature is 30-40 ℃.
2. The method according to claim 1, wherein the fermentation medium for the fermentation comprises the following components: 5-20g/L glucose, 5-20g/L peptone, 1-10g/L beef powder and 1-10g/L, NaH yeast powder2PO4 1-5 g/L、NaCl 1-5 g/L、MgSO4·7H2O 0.1-2 g/L。
3. The method according to claim 1, wherein the nanocellulose is added in the form of a nanocellulose dispersion.
4. The method according to claim 1, wherein the amount of nanocellulose to be added is controlled so that the final mass content of nanocellulose in the system is 0.5%.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN106309473A (en) * 2015-07-03 2017-01-11 格莱克生物科学公司 Polymer matrix compositions comprising a high concentration of bio-fermented sodium hyaluronate and uses thereof

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WO2008035372A2 (en) * 2006-07-06 2008-03-27 Reliance Life Sciences Pvt Ltd Efficient process for purification of high molecular weight hyaluronic acid
US20100137579A1 (en) * 2008-12-01 2010-06-03 Sustineo Biotechnology Co., Ltd. Process for purifying medical grade hyaluronic acid

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CN106309473A (en) * 2015-07-03 2017-01-11 格莱克生物科学公司 Polymer matrix compositions comprising a high concentration of bio-fermented sodium hyaluronate and uses thereof

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