CN114574403A - Method for preparing thermophilic thermus strain fermentation product by utilizing rice bran enzyme hydrolysate - Google Patents
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Abstract
The invention provides a method for preparing a thermophilic thermus strain fermentation product by fermenting rice bran enzyme hydrolysate. According to the invention, cheap rice bran enzyme hydrolysate is used as a thermophilic thermus inhabiting bacteria fermentation substrate, thermophilic thermus inhabiting bacteria fermentation can be carried out independently or saccharomyces cerevisiae is adopted for co-fermentation, the preparation cost of thermophilic thermus inhabiting bacteria fermentation products can be effectively reduced, the preparation method can be used for simultaneously preparing the intracellular and extracellular fermentation products of thermophilic thermus inhabiting bacteria, the fermentation products have high polysaccharide and amino acid contents, and the tyrosinase inhibition rate and the DPPH free radical scavenging rate can be improved. The intracellular and extracellular products synthesized by thermophilic thermus bacteria have biological activities of oxidation resistance, whitening and the like, and can be applied to the cosmetic industry, thereby realizing the comprehensive high-value utilization of the functional components of the rice bran and the thermophilic thermus bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, relates to a method for preparing a thermophilic thermus strain fermentation product by using rice bran enzyme hydrolysate.
Background
The fermentation product of Thermus thermophilus has photoprotection, and can be used for preventing or treating injury related to ultraviolet and infrared free radicals, and repairing skin injury. The fermentation product can not only reduce wrinkles to a large extent, improve skin integrity, deeply moisturize, nourish and improve elasticity, but also has antioxidant effect, and thus can be added to cosmetics as an anti-aging and moisturizing ingredient. At present, the thermophilic thermus strain fermentation product has more functions, but the production cost is higher, so the application of the thermophilic thermus strain fermentation product in cosmetics is limited.
China is one of the countries with the largest rice production in the world, and the annual output is about 2 hundred million tons. In China, rice is used as staple food in population exceeding 2/3, and the consumption of rice accounts for more than 6% of the total consumption of grain. The rice bran is a main byproduct in the process of processing the rice into rice, accounts for 6-8% of the weight of the rice, is mainly used as feed at present, has low added value, and needs to be explored for a high-value utilization way of the rice bran. So far, the prior art also uses rice bran as raw material for processing treatment, for example, chinese patent CN101575620A provides a pretreatment method using rice bran as fermentation raw material; patent CN110205349A discloses a method for preparing bacterial cellulose by fermentation of rice bran hydrolysate; in addition, Haokifang et al invented a method for preparing lactic acid by fermenting rice bran hydrolysate (research on L-lactic acid prepared by biological refining of Haokifang and rice bran, Shandong province, Shandong Fuxin Biotechnology GmbH, 2018-12-27.). Although rice bran has been used for high-value utilization in the prior art, few methods are available for processing and preparation using rice bran as a raw material, and no literature report on the preparation of a thermophilic thermus strain fermentation product using rice bran has been found.
At present, researches on preparation of fermentation products of thermus thermophilus are few, and Chinese patent CN 108517345A discloses a combined fermentation method of thermus thermophilus and microzyme, which adopts the thermus thermophilus to carry out secondary fermentation, and can improve some active ingredients of the thermus thermophilus; although the method can improve the active ingredients of the thermus thermophilus, the method needs strict control of acid-base conditions of fermentation, needs multiple times of enrichment culture and is complicated in process, and only the mixture of the fermentation products of the thermus thermophilus, namely the extracellular product of the thermus thermophilus is used as an effective functional ingredient, so that the prepared thermus thermophilus product does not fully play a role, and the synthetic culture medium used for the fermentation is high in cost. At present, the preparation of the thermophilic thermus bacteria fermentation product by utilizing rice bran is rarely reported, but aiming at the defects of the prior art, in order to more fully utilize the thermophilic thermus bacteria fermentation product, the development of a thermophilic thermus bacteria fermentation product with simplicity, low cost and high biological activity and a preparation method thereof are needed, and the thermophilic thermus bacteria fermentation product can be applied to large-scale production and preparation and can realize high-value utilization of the rice bran.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings in the prior art and provides a method for preparing a thermophilic thermus strain fermentation product by fermenting rice bran enzyme hydrolysate.
The invention aims to provide a method for preparing a thermophilic thermus strain fermentation product by utilizing rice bran enzyme hydrolysate.
The above purpose of the invention is realized by the following technical scheme:
in order to effectively reduce the preparation cost of the thermophilic thermus strain fermentation product, the invention researches a method for preparing the thermophilic thermus strain fermentation product by using rice bran enzyme hydrolysate as a fermentation substrate.
A method for preparing a thermus thermophilus fermentation product by utilizing rice bran enzyme hydrolysate comprises the following steps:
s1, performing diastatic enzyme hydrolysis on rice bran to obtain enzyme hydrolysate, performing solid-liquid separation on the enzyme hydrolysate, and taking supernate for sterilization for later use;
s2, taking the rice bran enzyme hydrolysate prepared in the step S1 as fermentation culture, and independently inoculating thermophilic thermus bacteria into a fermentation culture medium for fermentation culture or firstly inoculating saccharomyces cerevisiae for fermentation culture and then inoculating thermophilic thermus bacteria for co-fermentation culture;
s3, after fermentation is finished, carrying out solid-liquid separation, respectively collecting precipitates and fermentation liquor, wherein the fermentation liquor obtained by separation is a thermophilic thermus habitats fermentation extracellular product with biological activity;
s4, washing and re-suspending the precipitate separated in the step S3, then carrying out cell disruption treatment, and then carrying out solid-liquid separation to obtain supernatant, namely the thermophilic thermus strain fermentation intracellular product with biological activity.
Preferably, the rice bran used in step S1 is subjected to a gelatinization process.
Preferably, the amount of Thermus thermophilus inoculated in step S2 is 5-10%.
More preferably, the amount of Thermus thermophilus inoculated in step S2 is 10%.
Preferably, the conditions for separately inoculating thermus thermophilus for fermentation culture in step S2 are as follows: 50-75 ℃, 150-200 rpm, 12-48 h.
More preferably, the conditions for separately inoculating thermus thermophilus for fermentation culture in step S2 are as follows: 60 ℃, 150rpm, 18 h.
Preferably, the ratio of the usage amount of the saccharomyces cerevisiae and the thermus thermophilus adopted in the step S2 is 0.5-1: 1 to 2.
More preferably, the ratio of the amount of saccharomyces cerevisiae to thermus thermophilus is 1: 1.
preferably, when the saccharomyces cerevisiae is adopted for the co-fermentation in the step S2, the fermentation conditions of the inoculated saccharomyces cerevisiae are as follows: 25-35 ℃, 150-200 rpm, 12-24 hours; the fermentation conditions after inoculating the thermophilic thermus strain are as follows: 50-75 ℃, 150-200 rpm, 12-48 h.
More preferably, when the saccharomyces cerevisiae is adopted for the co-fermentation in the step S2, the fermentation conditions of the inoculated saccharomyces cerevisiae are as follows: at 30 ℃, 200rpm for 12-14 h; the fermentation conditions after inoculating the thermus thermophilus are as follows: 60 ℃, 150rpm, 18-24 h.
Preferably, the solid-liquid separation in step S1, step S3 and step S4 is performed by centrifugation or suction filtration.
Preferably, the method of cell disruption treatment in step S4 is ultrasonic disruption or other methods suitable for cell disruption.
More preferably, the conditions of the ultrasonication are: the power is 250-300W, and the time is 2-4 h.
The invention has the following beneficial effects:
the invention provides a method for preparing a thermophilic thermus strain fermentation product by fermenting rice bran enzyme hydrolysate, which adopts the rice bran enzyme hydrolysate as a fermentation substrate, can perform thermophilic thermus strain fermentation independently or perform co-fermentation by adopting saccharomyces cerevisiae, and can effectively reduce the preparation cost of the thermophilic thermus strain fermentation product. The intracellular product obtained by single-bacterium fermentation of thermophilic thermus strain has more bioactivity than extracellular product, better antioxidizing property, and high value and potential. The antioxidant capacity of extracellular products can be improved by carrying out the co-fermentation of the saccharomycetes and the thermus thermophilus, the prepared thermus thermophilus fermentation product has high polysaccharide and amino acid contents, and can also improve the tyrosinase inhibition rate and the DPPH free radical clearance rate, and the whitening effect is shown in the thermus thermophilus fermentation product prepared by the method.
The thermophilic thermus strain fermentation product prepared by the method has high antioxidant activity, and can be used for simultaneously preparing intracellular and extracellular fermentation products of thermophilic thermus strains, so that the utilization value of the thermophilic thermus strains is improved.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The following examples use the following media for the activation of yeasts and Thermus thermophilus:
thermus thermophilus activation medium g/L: peptone 3, glucose 0.2, anhydrous magnesium sulfate 0.35, potassium dihydrogen phosphate 0.12, ammonium sulfate 2, ferric chloride 0.05, sodium chloride 0.25, pH7.8, sealing and sterilizing (50 mL per bottle);
yeast activation medium g/L: peptone 3, glucose 0.2, anhydrous magnesium sulfate 0.35, potassium dihydrogen phosphate 0.12, ammonium sulfate 2, ferric chloride 0.05, sodium chloride 0.25, pH4.8, sealed and sterilized (50 mL per bottle).
The rice bran enzyme hydrolysate used in the following examples was obtained by hydrolyzing rice bran with saccharifying enzyme. Wherein the sugar content of the rice bran hydrolysate used for fermentation is about 500 mg/L.
The following examples employ the DPPH free radical scavenging test method: according to the proportion of 1: 1, mixing a sample to be detected with 0.1mmol/L DPPH 70% ethanol solution, shaking up, standing in the dark at room temperature for 30min, and measuring the light absorption value at 517 nm.
DPPH clearance (%) < 1- (A)1-A2)/A0×100％
In the formula: a. the1Adding a light absorption value of a DPPH ethanol solution to a sample to be detected;
A2adding the absorbance of the solvent to the sample;
A0the absorbance of the DPPH ethanol solution plus solvent.
EXAMPLE 1 fermentation of Thermus thermophilus Single bacterium to prepare intracellular and extracellular fermentation products
Gelatinizing rice bran, hydrolyzing with diastase to obtain enzyme hydrolysate, vacuum filtering, and steam sterilizing the supernatant. Thermus thermophilus was activated, inoculated into a fermentation medium (pH 7.8) containing a 10% inoculum size of a rice bran enzyme hydrolysate, subjected to fermentation culture, and cultured at 60 ℃ and 150rpm for 18 hours.
After fermentation, performing solid-liquid separation by centrifugation, and respectively collecting precipitate and fermentation liquor, wherein the fermentation liquor is extracellular product of thermophilic thermus strain. The DPPH free radical clearance test result shows that the DPPH free radical clearance of the fermentation liquid thermophilic thermus strain fermentation extracellular product reaches 46 percent, which shows that the thermophilic thermus strain fermentation extracellular product has the anti-oxidation capability.
Washing the precipitate obtained by the separation with deionized water, adding deionized water to re-suspend cells, crushing the cells for 2h by ultrasonic waves with the power of 300W, centrifuging after ultrasonic treatment, centrifuging to obtain supernatant, namely the thermophilic thermus thermophilus intracellular fermentation product, and determining the DPPH free radical clearance rate, wherein the DPPH free radical clearance rate of the thermophilic thermus thermophilus intracellular fermentation product is 65%, which shows that the thermophilic thermus thermophilus intracellular fermentation product has better oxidation resistance.
EXAMPLE 2 fermentation of Thermus thermophilus with Yeast in combination to prepare intracellular and extracellular fermentation products
Gelatinizing rice bran, hydrolyzing with diastase to obtain enzyme hydrolysate, vacuum filtering the enzyme hydrolysate, and steam sterilizing the supernatant. Activating yeast and Thermus thermophilus, inoculating yeast into rice bran enzyme hydrolysate (pH 7.8) at an inoculum size of 10%, fermenting, culturing at 30 deg.C for 14 hr at 200rpm, and adjusting pH of the fermentation broth to 7.8; then, Thermus thermophilus was inoculated at an inoculum size of 10%, and fermentation was continued at 60 ℃ and 150rpm for 18 hours.
And after the fermentation is finished, performing solid-liquid separation by centrifugation, and respectively collecting precipitates and fermentation liquor, wherein the fermentation liquor is a thermophilic thermus strain fermentation extracellular product, and a DPPH free radical clearance test result shows that the DPPH free radical clearance of the thermophilic thermus strain fermentation extracellular product in the fermentation liquor reaches 60%, which shows that the thermophilic thermus strain fermentation extracellular product has antioxidant capacity.
Washing the precipitate obtained by the separation with deionized water, adding deionized water to re-suspend cells, crushing the cells for 2h by ultrasonic waves with the power of 300W, centrifuging after ultrasonic treatment, and centrifuging to obtain supernatant, namely the thermophilic thermus thermophilus intracellular fermentation product, wherein the DPPH free radical clearance rate of the thermophilic thermus thermophilus intracellular fermentation product is determined to be 35%, which shows that the thermophilic thermus thermophilus intracellular fermentation product has better oxidation resistance.
Example 3 fermentation of Thermus thermophilus with Yeast in combination
Gelatinizing rice bran, hydrolyzing with diastase to obtain enzyme hydrolysate, vacuum filtering, adding 10g/L nitrogen source (yeast extract powder) into the enzyme hydrolysate, and steam sterilizing the supernatant. Respectively activating yeast and Thermus thermophilus, inoculating yeast into rice bran enzyme hydrolysate (pH 7.8) at an inoculum size of 10%, fermenting and culturing at 30 deg.C and 200rpm for 12 hr, and adjusting pH of the fermentation liquid to 7.8; then, Thermus thermophilus was inoculated at an inoculum size of 10%, fermentation culture was continued, and culture was carried out at 60 ℃ and 150rpm for 24 hours. And (4) carrying out solid-liquid separation after the fermentation is finished to obtain fermentation liquor.
The fermentation liquor is taken for determination, and the result shows that the polysaccharide content is 736 mug/mL, the amino acid content is 2.58g/L, the tyrosinase inhibition rate is 43 percent, and the DPPH free radical clearance rate is 54.7 percent.
Comparative example 1
Respectively activating yeast and Thermus thermophilus, inoculating yeast into prepared fermentation culture medium (pH 4.8 yeast activation culture medium) at an inoculum size of 10%, performing fermentation culture, culturing at 30 deg.C and 200rpm for 12 hr, and adjusting pH of the fermentation broth to 7.8; then, Thermus thermophilus was inoculated at an inoculum size of 10%, fermentation culture was continued, and culture was carried out at 60 ℃ and 150rpm for 24 hours. And (4) carrying out solid-liquid separation after the fermentation is finished to obtain fermentation liquor.
The fermentation liquor is taken for measurement, and the result shows that the polysaccharide content is 51.2 mu g/mL, the amino acid content is 1.81g/L, the tyrosinase inhibition rate is 7 percent, and the DPPH free radical clearance rate is 53.6 percent.
The results of the single-bacterium fermentation and the co-fermentation of the rice bran hydrolysate in the embodiment 1 and the embodiment 2 respectively show that the DPPH free radical clearance rate of the co-fermented extracellular product of the thermophilic thermus strain fermentation is 60 percent, and the oxidation resistance of the co-fermented extracellular product of the thermophilic thermus strain fermentation is improved by 14 percent compared with that of the single-bacterium fermented extracellular product of the thermophilic thermus strain fermentation. It is known that the combination of yeast and Thermus thermophilus, when fermented, can increase the activity of the extracellular fermentation product. And the rice bran hydrolysate is adopted for co-fermentation, so that the acid-base conditions of fermentation do not need to be strictly controlled, and the enrichment culture is also not needed.
In the embodiment 3 of the invention, the nitrogen source is added into the fermentation medium, and compared with the embodiment 2, the nitrogen source is added, so that the nitrogen source has no influence on mixed fermentation; compared with the comparative example 1, the example 3 has the advantages that the polysaccharide content is obviously higher than that of the comparative example 1, the amino acid content is also higher than that of the comparative example 1, the tyrosinase inhibition rate is also obviously higher than that of the comparative example 1, and the stronger the tyrosinase inhibition effect is, the better the whitening effect is; therefore, the rice bran enzyme hydrolysate can be used for fermentation, so that the contents of polysaccharide and amino acid can be increased, and the tyrosinase inhibition rate and the DPPH free radical clearance rate can be increased.
In conclusion, the activities of fermentation products obtained by different fermentation combinations of the thermophilic thermus strain are different, intracellular products obtained by single-strain fermentation of the thermophilic thermus strain have more biological activity than extracellular products, and the oxidation resistance of the intracellular products is better; when the co-fermentation is carried out, the clearance rate of the extracellular product of the thermus thermophilus is higher than that of DPPH free radical of the intracellular product, which shows that the co-fermentation can improve the oxidation resistance of the extracellular product. The method for preparing the thermophilic thermus strain fermentation product by fermenting the rice bran enzyme hydrolysate provided by the invention not only can obtain intracellular fermentation products with higher activity by single strain fermentation, but also can improve the biological activity of the extracellular fermentation products by co-fermentation.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing a thermus thermophilus fermentation product by utilizing rice bran enzyme hydrolysate is characterized by comprising the following steps:
s1, performing diastatic enzyme hydrolysis on rice bran to obtain enzyme hydrolysate, performing solid-liquid separation on the enzyme hydrolysate, and taking supernate for sterilization for later use;
s2, taking the rice bran enzyme hydrolysate prepared in the step S1 as a fermentation culture medium, and independently inoculating thermophilic thermus bacteria into the fermentation culture medium for fermentation culture or firstly inoculating saccharomyces cerevisiae for fermentation culture and then inoculating thermophilic thermus bacteria for co-fermentation culture;
s3, after fermentation is finished, carrying out solid-liquid separation, respectively collecting precipitates and fermentation liquor, wherein the fermentation liquor obtained by separation is a thermophilic thermus habitats fermentation extracellular product with biological activity;
s4, washing and re-suspending the precipitate separated in the step S3, then carrying out cell disruption treatment, and then carrying out solid-liquid separation to obtain supernatant, namely the thermophilic thermus strain fermentation intracellular product with biological activity.
2. The method of claim 1, wherein the rice bran used in step S1 is subjected to gelatinization.
3. The method according to claim 1, wherein the amount of Thermus thermophilus inoculated in step S2 is 5-10%.
4. The method according to claim 3, wherein the conditions for separately inoculating Thermus thermophilus for fermentation culture in step S2 are as follows: 50-75 ℃, 150-200 rpm, 12-48 h.
5. The method according to claim 1, wherein the inoculation amount ratio of Saccharomyces cerevisiae to Thermus thermophilus used in step S2 is 0.5-1: 1 to 2.
6. The method according to claim 5, wherein the ratio of the inoculum size of Saccharomyces cerevisiae to Thermus thermophilus is 1: 1.
7. the method according to claim 6, wherein in the step S2, when Saccharomyces cerevisiae is used for co-fermentation, the fermentation conditions of the inoculated Saccharomyces cerevisiae are as follows: at 25-35 ℃, 150-200 rpm for 12-24 h; the fermentation conditions after inoculating the thermophilic thermus strain are as follows: 50-75 ℃, 150-200 rpm, 12-48 h.
8. The method of claim 7, wherein the solid-liquid separation in step S1, step S3 and step S4 is performed by centrifugation or suction filtration.
9. The method of claim 8, wherein the cell disruption treatment in step S4 is ultrasonic disruption or other methods suitable for cell disruption.
10. The method of claim 9, wherein the conditions for the ultrasonication are as follows: the time is 2-4 h, and the power is 250-300W.
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| CN116497077A (en) * | 2023-05-12 | 2023-07-28 | 广东药科大学 | Method for preparing thermophilic thermus fermentation product by using tremella extract |
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| 祁红兵;陈钧;何佳;徐骅;熊郁云;: "纳豆芽孢杆菌发酵米糠上清液的抗氧化功能研究", 食品科学, no. 03 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116497077A (en) * | 2023-05-12 | 2023-07-28 | 广东药科大学 | Method for preparing thermophilic thermus fermentation product by using tremella extract |
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