CN118064277A - Lucid ganoderma and probiotics coupled fermentation product, fermentation method and application - Google Patents
Lucid ganoderma and probiotics coupled fermentation product, fermentation method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial cultivation, and particularly relates to a ganoderma lucidum and probiotics coupled fermentation product, a fermentation method and application. The invention realizes the utilization of active ingredients in the supernatant of the submerged fermentation of the ganoderma lucidum by probiotics, reduces the waste of nutrient ingredients such as monosaccharide, polysaccharide, nitrogen source and the like in the supernatant of the submerged fermentation of the ganoderma lucidum, and simultaneously obtains a plurality of fermentation products of strains which are compounded with two effects of the ganoderma lucidum and the probiotics simultaneously, such as extracellular polysaccharide, ganoderma lucidum triterpene, organic acid, mycoprotein and the like, and substances transformed by interaction thereof, thereby obviously improving the biological antioxidation effect, delaying aging, regulating the ecological environment of skin and enhancing the immunity. Meanwhile, the times and energy consumption of thalli centrifugation, fermentation tank sterilization and concentration and extraction of fermentation supernatant are reduced when two thalli are respectively fermented, and the high-temperature decomposition of functional products is avoided when the fermentation supernatant is concentrated and recovered for a long time.
Description
Technical Field
The invention belongs to the technical field of microbial cultivation, and particularly relates to a ganoderma lucidum and probiotics coupled fermentation product, a fermentation method and application.
Background
Ganoderma (Ganoderma) belongs to Polyporaceae fungi of Basidiomycetes, is a traditional and rare Chinese medicine, has abundant chemical components, contains polysaccharide, triterpene, mineral substances, microelements, flavone, polyphenol, etc., and has effects of resisting oxidation, delaying aging, promoting skin metabolism, enhancing immunity, etc. The common form of ganoderma lucidum is a highly lignified fruiting body (fruiting bodies), seeds released by ejection in the later stage of development are collected and are in powder form, namely spore powder (spores), spores germinate to form mycelium (mycelium), the mycelium continues to develop and mature to form the fruiting body, and the 3 forms are the growth cycle of ganoderma lucidum.
Ganoderan and ganoderan are widely regarded as main bioactive components in ganoderma lucidum, and have biological activities of antioxidation, immunoregulation, anti-tumor, liver protection, blood sugar reduction, cardiovascular system and neuroprotection, etc. Ganoderma triterpene and polysaccharide can be extracted from fruiting body, spore powder, mycelium and fermentation broth of Ganoderma. Wherein, mycelium polysaccharide produced by liquid fermentation is intracellular polysaccharide, fermentation broth polysaccharide is extracellular polysaccharide, and both are effective polysaccharides. At present, the liquid state fermentation is gradually replaced by artificial cultivation as a main method for extracting ganoderan and triterpene, and the research on the liquid state submerged fermentation of ganoderma is mostly the research on the optimal cultivation method of ganoderma mycelia, and the research on comprehensive application and deep development of fermentation products are rarely related.
The lysate of probiotics such as bifidobacterium, lactobacillus and other thallus contains rich polysaccharide, polypeptide and organic acid nutrient components, and can be applied to various health foods and cosmetics for regulating skin ecological environment, intestinal flora and organism nutrient balance. In addition, research shows that the probiotics metabolism polysaccharide can produce various beneficial metabolites such as lactic acid, short chain fatty acid and the like, and has obvious probiotics effect. Meanwhile, the probiotics and the ganoderma lucidum co-ferment can mutually utilize the generated polysaccharide, active enzyme and the like to promote the further conversion or decomposition of ganoderma lucidum fermentation products, so that new active substances are obtained, and the prebiotic activity and bioavailability of the active substances are improved. Although the prior art relates to the application of probiotics to ganoderma lucidum processing, the advanced development and research of the primary processing products of ganoderma lucidum fruiting bodies or ganoderma lucidum fermentation mycelia are mostly carried out, but the utilization of ganoderma lucidum mycelia fermentation liquor is not comprehensive enough, so that the waste of active ingredients in supernatant after the fermentation of the mycelia is caused.
For example, patent CN114209617a is a yeast fermented ganoderma lucidum extract, a preparation method and application thereof, CN108004282a is a method for preparing ganoderma lucidum fruiting body ferment, CN111973498A is a ganoderma lucidum ferment, a preparation method and application thereof, CN113768831a is a method for preparing ganoderma lucidum secondary fermentation liquid, fermentation liquid and application thereof, although the utilization efficiency of intracellular polysaccharide, fruiting body or mycelium of ganoderma lucidum is promoted by the action of probiotics on ganoderma lucidum fermentation, the utilization of ganoderma lucidum liquid submerged fermentation supernatant is still lacking. The wider the application of mycelium obtained by submerged fermentation of ganoderma lucidum liquid, the larger the yield requirement, and the more fermentation supernatant and culture waste are wasted.
In the patent of utilizing the supernatant fluid and the culture of ganoderma lucidum, CN114044809A is an antioxidant active peptide derived from ganoderma lucidum and application thereof, CN101455274A is a method for preparing feed and feed additive containing ganoderma lucidum active ingredients by using ganoderma lucidum liquid fermentation liquor, CN111053711A is a ganoderma lucidum probiotics composite fermentation product, a preparation method and application thereof, and the like, and the invention is only the utilization of the supernatant fluid after separation, and does not relate to secondary development and efficacy enhancement of the supernatant fluid of ganoderma lucidum fermentation. The development of the ganoderma lucidum fermentation supernatant fluid does not relate to the interaction of ganoderma lucidum mycelia and probiotics strains, so that the product has low nutrition composition richness, the efficacy of the ganoderma lucidum fermentation supernatant fluid cannot be further improved, and the high-value comprehensive utilization of ganoderma lucidum fermentation products cannot be realized. While patent CN113502232a provides a method for preparing mixed seed liquid of ganoderma mycelia and hericium erinaceus mycelia and performing mixed fermentation, although the improvement of the utilization rate of ganoderma fermentation media and the generation of interaction products are realized at the same time, both are large fungi, carbon and nitrogen sources are required to be similar, so that the utilization of the media is insufficient, the similarity of active ingredients is high, the nutrition complementation and the mutual synergistic interaction among the mycelia are limited, and the improvement of the effect of fermentation media is limited.
In order to achieve the purpose of interaction and synergistic effect of ganoderma lucidum and probiotics, a preparation method of ganoderma lucidum secondary fermentation liquid, fermentation liquid and application thereof of patent CN113768831A are characterized in that ganoderma lucidum submerged liquid fermentation products are filtered, fermentation mycelium is subjected to enzymolysis, lactobacillus fermentation and saccharomycetes secondary fermentation after filtrate removal, and the obtained ganoderma lucidum secondary fermentation liquid is rich in micromolecular functional substances generated by interaction of ganoderma lucidum mycelium and probiotics, but still causes waste of ganoderma lucidum fermentation supernatant, so that a plurality of active ingredients and nutrient substances such as extracellular polysaccharide and the like are lost. In addition, the method needs to be subjected to operations such as twice centrifugal recovery, twice fermentation tank sterilization and the like, has complex steps, and is high in energy consumption and preparation cost when applied to industrialization.
Therefore, it is needed to provide a coupled fermentation method of ganoderma lucidum and probiotics, which is simple to operate and low in energy consumption, and is suitable for industrial production, so that the utilization value of ganoderma lucidum fermentation liquid is improved, and meanwhile, various thallus components such as ganoderma lucidum and probiotics are obtained. Meanwhile, the coupled ferment rich in the interaction product of the lucid ganoderma and the probiotics and the composite thallus of the lucid ganoderma and the probiotics are produced.
Disclosure of Invention
The invention aims to provide a ganoderma lucidum and probiotics coupled fermentation product, a fermentation method and application, which utilize the culture components (ganoderma lucidum fermentation residual nutrient components, ganoderma lucidum extracellular polysaccharide, triterpene, trace elements and the like) at the later fermentation stage of ganoderma lucidum as prebiotics, properly supplement necessary nutrition to carry out probiotics coupled fermentation, obtain a novel raw material of cosmetics and health foods rich in a plurality of nutrient components such as polysaccharide, mineral substances, trace elements, flavone, polyphenol, polypeptide, organic acid and the like, and the raw material has various effects of resisting oxidation, regulating the ecological environment of skin, enhancing immunity and the like, simultaneously reduces the operation procedures such as centrifugation, transfer tank and the like, reduces energy loss, reduces the waste of ganoderma lucidum fermentation supernatant and provides a novel application direction for the raw material.
Based on the above object, the application provides a coupled fermentation method of ganoderma lucidum and probiotics, comprising the following steps: (1) Activating, culturing, expanding culturing and fermenting the ganoderma lucidum to obtain a ganoderma lucidum submerged fermentation product; (2) Directly inoculating probiotics into the submerged fermentation product of ganoderma lucidum to perform coupling fermentation, and simultaneously obtaining coupled fermentation product thalli and fermentation supernatant; (3) And freeze-drying, vacuum drying under reduced pressure or spray drying the thallus layer and supernatant after coupling fermentation for standby.
Specifically, the method for obtaining the ganoderma lucidum submerged fermentation product comprises the following steps: inoculating Ganoderma to PDA slant culture medium, culturing at 28deg.C in dark for 7 days, inoculating slant strain to Ganoderma liquid culture medium, culturing at 28deg.C at 220r/min for 3 days under natural pH, transferring 5% of the inoculating amount of liquid strain to shake flask, expanding culturing for 5 days, inoculating 5% of the inoculating amount to 100L fermenter, stirring at 160r/min, and regulating air vent valve to maintain dissolved oxygen content at 40% and 28deg.C for 4 days.
Specifically, before probiotics are inoculated for coupling fermentation, a supplementary nutrient medium is added into the ganoderma lucidum submerged fermentation.
More specifically, the formula of the nutrient supplement medium provided by the invention is as follows: adding 50mL of supplementary culture medium into every 1000mL of Ganoderma lucidum liquid fermentation; every 50mL of the supplementary culture medium contains 5g of fructo-oligosaccharide, 3g of glucose, 5g of rice peptide and 2g of folic acid.
Further, the probiotics are lactobacillus or bifidobacterium.
Preferably, the lactobacillus of the present invention is lactobacillus rhamnosus.
Preferably, the bifidobacterium of the present invention is bifidobacterium longum.
Specifically, the coupling fermentation culture equipment can adjust stirring speed and ventilation, control dissolved oxygen, and perform aerobic and anaerobic fermentation simultaneously. The culture conditions are as follows: after the ganoderma lucidum is fermented for 4 days, the temperature of a fermentation tank is regulated to 37 ℃, a ventilation valve is closed, the stirring speed is regulated to 50r/min, the supplementary nutrient medium is added, probiotics are inoculated into the fermentation tank, the inoculation amount of the probiotics is 2%, the fermentation tank is cultured for 48 hours, and the pH is regulated by utilizing NaHCO 3 for 24 hours before the culture, so that the pH is maintained near 6.
Specifically, the centrifugation condition is 5000-10000 r/min, and the centrifugation is 15-20 min.
Specifically, the drying in the invention is freeze drying, reduced pressure vacuum drying or spray drying. On the other hand, the coupled fermentation product of the interaction product of the ganoderma lucidum and the probiotics and the composite thallus of the ganoderma lucidum and the probiotics are obtained through the coupled fermentation method of the ganoderma lucidum and the probiotics.
In a third aspect, the invention also provides a coupled fermentation method of ganoderma lucidum and probiotics, a coupled fermentation product obtained by the method, and application of the ganoderma lucidum and probiotics composite thalli in cosmetics and health foods.
Compared with the prior art, the invention has the following beneficial effects or advantages:
1. The invention utilizes the culture components (residual nutrient components of ganoderma lucidum fermentation, extracellular polysaccharide, triterpene, trace elements and the like) of ganoderma lucidum at the later stage of fermentation as prebiotics, properly supplements necessary nutrition to carry out coupled fermentation of probiotics, converts part of the components into active substances such as thalli, extracellular polysaccharide, organic acid, probiotic polypeptide, short chain fatty acid and the like of the probiotics, and generates new nutrient substances by the interaction of ganoderma lucidum and the fermentation of the probiotics. The process not only improves the diversity and the efficacy of fermentation functional products, but also avoids the waste of the components of the fermentation supernatant of the ganoderma lucidum cells.
2. The invention captures the functional components in the lucid ganoderma fermentation supernatant by utilizing probiotics, and obtains lucid ganoderma probiotics composite thalli and coupled fermentation supernatant by simple operation and one-time centrifugation, wherein the supernatant contains lucid ganoderma extracellular polysaccharide, probiotic extracellular polysaccharide, triterpene, organic acid, probiotic polypeptide, short chain fatty acid and other lucid ganoderma and probiotic extracellular functional components. The process reduces secondary centrifugation and secondary sterilization of fermentation tank required by fermenting Ganoderma and probiotic respectively, and solves the problems of high energy consumption and high temperature decomposition of product during drying and recovering process of fermentation supernatant of Ganoderma and probiotic respectively.
3. According to the invention, the lucid ganoderma and the probiotics are subjected to coupled fermentation to obtain substances produced by respective fermentation of the lucid ganoderma and the probiotics and products obtained by interaction conversion of the substances, and fermentation products such as polysaccharide, protein, small molecular compounds and the like are metabolized or converted through interaction to generate new functional components such as polysaccharide molecular structure change, new functional polypeptide generation, rhamnose and xylose content change, and organic acids such as lactic acid, citric acid and the like appear.
4. Polysaccharides, triterpenes, flavones, polyphenols and the like in the ganoderma lucidum have the effects of resisting oxidation, promoting skin metabolism, reducing oxidative stress of the organism, improving the immunity of the organism and the like; the probiotics such as bifidobacterium, lactobacillus and the like contain rich polysaccharide, polypeptide and organic acid nutrition components, can regulate the balance of skin flora, and have the effects of resisting bacteria, resisting inflammation and the like. The invention obtains the cosmetics and health food functional raw materials rich in ganoderma lucidum, probiotics and various nutritional ingredients generated by the interaction of the ganoderma lucidum and the probiotics through one-time coupling fermentation.
5. The invention establishes a set of ganoderma lucidum mycelium and probiotics coupled fermentation process by utilizing the difference of fermentation characteristics of ganoderma lucidum mycelium and probiotics, and is used for preparing novel raw materials of cosmetics and health foods rich in multiple nutritional ingredients such as polysaccharide, flavone, polyphenol, polypeptide, organic acid, mineral substances, trace elements and the like.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods and the detection methods in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless specified, are commercially available; the index data are all conventional measurement methods unless specified.
The strains used in the examples below were all deposited in the institute of microorganisms, shaanxi province.
Example 1 coupled fermentation method of Ganoderma lucidum and probiotic
The ganoderma lucidum of this example is selected from ganoderma lucidum (Ganoderma lucidum. Karst) deposited in the institute of microbiology, shaanxi, accession number MU015.
Inoculating the above Ganoderma to PDA slant culture medium (20% potato juice 1L, glucose 20g, mgSO 4 1.5g,KH2PO4 3g,VB1 mg, agar 20g,20% potato juice preparation method: potato is cleaned and peeled, cutting 200g into small pieces, adding 1000mL of water, boiling for 20min, filtering to remove potato pieces, supplementing 1000mL of filtrate with distilled water), and culturing at 28deg.C in dark for 7 days for activation. Inoculating slant strain of Ganoderma lucidum into Ganoderma lucidum liquid culture medium (glucose 20g, corn flour 20g, bean cake flour 20g, KH 2PO4 10g,MgSO4 0.5.5 g, constant volume of distilled water to 1000 mL), rotating shaking table 220r/min under natural pH environment, liquid culturing at 28deg.C for 3 days, transferring 5% inoculum size of liquid strain to shaking bottle, expanding culturing for 5 days, inoculating 5% inoculum size into 100L fermenter, stirring at 160r/min, regulating vent valve to maintain dissolved oxygen amount at 40%, 28deg.C, fermenting for 4 days to obtain Ganoderma lucidum submerged fermentation product containing Ganoderma lucidum submerged fermentation supernatant and Ganoderma mycelia, taking 50mL Ganoderma lucidum submerged fermentation product to detect effective components, leaving most of Ganoderma lucidum submerged fermentation product in fermenter, adding supplementary nutrient medium, adding 50mL supplementary culture medium (5 g fructo-oligosaccharide, 3g glucose, 5g rice peptide, and folic acid 2 g) per 1000mL Ganoderma lucidum liquid fermentation product.
After the supplementary nutrient medium is prepared and added, naHCO 3 is used for regulating the PH to 5, lactobacillus rhamnosus SQ005 preserved in the institute of microbiology and microbiology of Shaanxi is inoculated to carry out coupling fermentation with ganoderma lucidum, the culture condition is 37 ℃, the ventilation valve is closed for 48 hours, and the stirring speed is regulated to 50r/min.
And centrifuging the coupled fermentation product under the conditions of 5000-10000 r/min and 15-20 min. The ganoderma lucidum probiotics coupled fermentation product thalli and fermentation supernatant are obtained, a small amount of the ganoderma lucidum probiotics coupled fermentation product thalli and the fermentation supernatant are respectively used for component detection, and the rest coupled fermentation product thalli of the embodiment are processed by freeze drying or reduced pressure vacuum drying, so that the composite thalli dry powder is obtained. And processing the rest coupled fermentation supernatant by adopting spray drying or reduced pressure vacuum drying to obtain the ganoderma lucidum and probiotics coupled fermentation supernatant dry powder.
Example 2 coupled fermentation method of Ganoderma lucidum and probiotic
A method for coupling fermentation of ganoderma lucidum and probiotics, which is similar to example 1, is characterized in that after the preparation of the nutrient supplement culture medium of example 1 and the addition of NaHCO 3 to adjust the PH to 5, lactobacillus rhamnosus (accession number: SQ 005) and bifidobacterium longum (accession number: SQ 0025) preserved in the institute of microorganisms of Shaanxi province are inoculated simultaneously for coupling fermentation with ganoderma lucidum.
Example 3 Strain selection experiments
In order to optimize the strain, proper strain is selected to perform coupling fermentation treatment on the ganoderma submerged fermentation product so as to obtain more effective components, and the effective components in the ganoderma submerged fermentation product are utilized to the maximum extent, so that the consumption of the supplementary nutrition is reduced. The species selected in this example include Lactobacillus rhamnosus SQ005, lactobacillus acidophilus R0047, lactobacillus casei R0025, lactobacillus reuteri R0005, lactobacillus plantarum R0069 and Bifidobacterium longum SQ0025 deposited in the Shaanxi institute of microbiology. The coupled fermentation was performed in the manner of example 1, and the activity of the strain in the submerged fermentation of ganoderma lucidum and the consumption of nutrients were determined by determining the time to reach the maximum viable count of the strain, the viable count of the probiotic, the dry weight of the cells of the coupled fermentation, and the residual sugar of the fermentation supernatant after adding the supplemental medium to 1000mL of the submerged fermentation of ganoderma lucidum.
(1) The number of viable bacteria of the coupled fermentation probiotics is measured by adopting a plate counting method, and the measurement is carried out every 4 hours.
Experiments show that the effective viable count of lactobacillus rhamnosus SQ005, lactobacillus acidophilus R0047, lactobacillus casei R0025, lactobacillus reuteri R0005, lactobacillus plantarum R0069 and bifidobacterium longum SQ0025 is up to 2.9×108CFU/mL,8.1×107CFU/mL,6.9×107CFU/mL,7.2×107CFU/mL,1.1×108CFU/mL and 2.7X10- 8 CFU/mL respectively, and the time for achieving the maximum viable count is 12h,16h,12 h and 12h respectively. Compared with other strains of lactobacillus rhamnosus SQ005, the lactobacillus rhamnosus SQ005 has higher activity, faster growth and better coupling property.
Compared with other strains, the effective viable count of the combined bacterium 1 (lactobacillus rhamnosus and bifidobacterium longum SQ0025 with the preservation number of SQ005 of Shaanxi province microorganism research institute) is highest, and reaches more than 3.3X10 8 CFU/mL, and the maximum viable count can be reached within 12 hours.
The maximum viable count of the combined bacterium 2 (Lactobacillus acidophilus and Bifidobacterium longum SQ0025 with accession numbers R0047 of Shaanxi province microorganism research institute) fermented for 16 hours can reach more than 1.9X10 8 CFU/mL, which shows that the probiotics have low activity, slow growth and poor coupling property compared with the environment of the invention of example 2 (the combined bacterium 1).
Other combinations, including Lactobacillus casei R0025, lactobacillus reuteri R0005 and Lactobacillus plantarum R0069 deposited in Shaanxi province institute of microbiology, were tested separately, and found that the number of viable bacteria in the bacterial suspension could only reach 8.5X10 7 CFU/mL or more, and the maximum viable bacteria could be reached 16 hours, which is less active, slow growing and unfavorable for coupling than the probiotic bacteria of example 2 (combination bacteria 1) of the present invention.
(2) And weighing the mass of the composite bacterial dry powder, and evaluating the bacterial dry weight of the coupled fermentation product. Experiments show that lactobacillus rhamnosus SQ005, lactobacillus acidophilus R0047, lactobacillus casei R0025, lactobacillus reuteri R0005, lactobacillus plantarum R0069 and bifidobacterium longum SQ0025 are respectively coupled with ganoderma lucidum, and the highest dry weight of the composite bacterial body obtained by fermenting is lactobacillus rhamnosus SQ005, which is 23.5g/L; the dry weight of the composite bacterial bodies obtained by the composite bacterial bodies 1 and 2 is 25.6g/L and 22.3g/L respectively, other combinations, including lactobacillus casei R0025, lactobacillus reuteri R0005 and lactobacillus plantarum R0069 which are preserved in the institute of microorganisms of Shaanxi province are respectively added and tested, and the dry weight of the coupling fermentation bacterial bodies of other composite bacterial strains is 20.8g/L at most, compared with the growth activity and the coupling effect of the composite bacterial body in the ganoderma lucidum submerged fermentation product in the embodiment 2 (the composite bacterial body 1) of the invention are poorer.
(3) The mass concentration of residual glucose in the supernatant after the submerged fermentation of the ganoderma lucidum and the coupling fermentation of the submerged fermentation and different combined bacteria is measured by a 3, 5-dinitrosalicylic acid colorimetric method, and the fact that the content of glucose in the lactobacillus rhamnosus fermentation supernatant in the supernatant after the coupling fermentation of the strain and the ganoderma lucidum is respectively lower is 3.24g/L is found; the glucose content of the fermentation supernatant of the combined bacterium 1 is the lowest and is 2.01g/L, the glucose content of the supernatant of the submerged fermentation of the original ganoderma lucidum is 8.35g/L, the glucose content of the supernatant of the combined bacterium 2 participating in the coupling fermentation is 3.94g/L, other combinations, including lactobacillus casei R0025, lactobacillus reuteri R0005 and lactobacillus plantarum R0069 which are preserved in the institute of microorganisms of Shaanxi province are respectively added to the test, and the glucose content of the supernatant of the coupling fermentation participated by other combined strains is found to be the lowest and is 4.11g/L, so that the invention can effectively utilize the glucose in the supernatant of the submerged fermentation of the original ganoderma lucidum under the coupling action of probiotics.
Example 4 Medium selection experiments
The different culture mediums supplement different nutrition in the ganoderma lucidum submerged fermentation products, so that the obtained fermentation products have different activities after full fermentation, and the specific formula is as follows:
supplementing nutrient medium # 1: adding 50mL of supplementary culture medium (containing fructo-oligosaccharide 5g, glucose 3g, rice peptide 5g, folic acid 2 g) into every 1000mL of Ganoderma submerged fermentation;
Supplementing nutrient medium # 2: adding 50mL of supplementary culture medium (containing fructo-oligosaccharide 5g, glucose 3g, corn peptide 5g, folic acid 2 g) into 1000mL of submerged fermentation;
supplementing nutrient medium 3#: 50mL of supplementary culture medium (the culture medium contains 5g of fructo-oligosaccharide, 3g of glucose, 5g of peptone and 2PO4 500mg,MgSO4200mg,VB1 mg of KH) is added into each 1000mL of ganoderma submerged fermentation;
nutrient medium supplement No. 4: adding 50mL of supplementary culture medium (the culture medium contains fructo-oligosaccharide 5g, glucose 3g, yeast powder 5g, KH 2PO4500mg,MgSO4200mg,VB1 mg) into every 1000mL of submerged fermentation of Ganoderma;
supplementing nutrient medium 5#: adding 50mL of supplementary culture medium (the culture medium contains 5g of glucose, 3g of tomato powder, 5g of yeast powder, 5g of beef powder, 2PO4 mg of KH 2PO4 mg, 200mg of Tween-80 and 500mg of sodium acetate) into every 1000mL of ganoderma submerged fermentation;
Supplementary nutrient medium # 6: 50mL of a supplementary medium (the medium contains 5g of glucose, 3g of sucrose, 5g of bean cake powder, 1g of peptone, 1g of yeast powder and 2PO4500mg,MgSO4 200mg,VB1 mg of KH) is added into each 1000mL of ganoderma submerged fermentation.
(1) The same coupled fermentation method of ganoderma lucidum and probiotics as in example 1 is adopted, the complementary nutrition culture medium is replaced, the coupled fermentation of ganoderma lucidum and probiotics (lactobacillus rhamnosus and lactobacillus longum and bifidobacterium longum) is respectively carried out, and the antioxidant activity of the fermentation supernatant is verified by detecting the DPPH free radical scavenging capacity of the fermentation supernatant. Taking 1mL of each of the coupled fermentation supernatant of different supplementing nutrient media and the coupled fermentation supernatant of the direct coupled fermentation non-supplementing nutrient media, and uniformly mixing with 1mL of 0.2mmol/L DPPH ethanol solution (sample tube to be tested); taking 1mL of absolute ethyl alcohol and respectively mixing the absolute ethyl alcohol with 1.0mL of fermentation supernatant (control tube); 1mL of absolute ethyl alcohol and 1mL of 0.2mmol/L DPPH ethanol solution are uniformly mixed (blank tube), and the light absorption value is measured at the wavelength of 517nm after the reaction is carried out at room temperature and in a dark place, wherein the calculation formula is as follows:
Clearance (%) = [1- (a i–Ax)/A0 ] ×100% formula, a i is absorbance of a sample tube to be measured, a x is absorbance of a control tube, and a 0 is absorbance of a blank tube.
(2) The same ganoderma lucidum and probiotics coupled fermentation method as in example 1 is adopted, the complementary nutrition culture mediums are replaced, the coupled fermentation is carried out respectively, after the different complementary nutrition culture mediums are measured by adopting a plate counting method, the maximum viable count and the corresponding time of inoculated probiotics (lactobacillus rhamnosus and the combination of lactobacillus longum) can be achieved, and meanwhile, the dry weight of the coupled ferment composite bacterial body is measured.
TABLE 1 determination of fermentation product index under coupled nutrient medium supplementation conditions
It can be seen from Table 1 whether the supplementation of the nutrient medium and its formulation has a major influence on the antioxidant activity of the final fermentation product. Experiments show that the coupled fermentation supernatant of the supplemented nutrient medium 1# has highest antioxidant activity, the DPPH free radical clearance of the coupled fermentation of ganoderma lucidum and lactobacillus rhamnosus and the coupled fermentation of ganoderma lucidum and combined bacteria (lactobacillus rhamnosus and bifidobacterium longum) respectively reach 88.45% and 91.07%, the viable count of bacterial suspension respectively reaches 2.91×10 8 CFU/mL and 3.32×10 8 CFU/mL, the maximum viable count can be reached within 12 hours, and the dry weight of the coupled fermentation product thallus respectively reaches 20.46g/L and 25.57 g/L.
Comparative example 1 Ganoderma lucidum submerged fermentation method
A method for deep fermentation of Ganoderma lucidum comprises inoculating Ganoderma lucidum of example 2 to PDA slant culture medium (20% potato juice 1L, glucose 20g, mgSO 4 1.5g,KH2PO4 3g,VB1 mg, agar 20g,20% potato juice preparation method: cleaning potato, peeling, cutting 200g into small pieces, adding 1000mL of water, boiling for 20min, filtering to remove potato pieces, supplementing 1000mL of filtrate with distilled water), and culturing at 28deg.C in dark place for 7 days for activation. Inoculating slant strain of Ganoderma into Ganoderma liquid culture medium (glucose 20g, corn flour 20g, bean cake flour 20g, KH 2PO4 10g,MgSO4 0.5.5 g, constant volume of distilled water to 1000 mL), rotating shaking table 220r/min under natural pH environment, liquid culturing at 28deg.C for 3 days, transferring 5% inoculum size of liquid strain to shaking flask, expanding culturing for 5 days, inoculating 5% inoculum size into 100L fermenter, stirring at 160r/min, regulating ventilation valve to maintain dissolved oxygen content at 40%, 28deg.C, fermenting for 4 days to obtain Ganoderma submerged fermentation supernatant, centrifuging at 8000rpm for 10min to obtain Ganoderma mycelia.
Comparative example 2 Ganoderma mycelia and probiotic composite fermentation method
A method for composite fermentation of ganoderma lucidum mycelia and probiotics, which is the same as that of example 2, is characterized in that the ganoderma lucidum submerged fermentation product obtained in example 2 is centrifuged at 5000r/min for 20min to obtain ganoderma lucidum fermentation mycelia, and then the ganoderma lucidum fermentation mycelia are transferred to a fermentation tank, and then 50mL of supplementary culture medium (the culture medium contains 5g of fructo-oligosaccharide, 3g of glucose, 5g of rice peptide and 2g of folic acid) is added, and after PH adjustment, probiotics are inoculated for composite fermentation.
Comparative example 3 Ganoderma lucidum supernatant and probiotic composite fermentation method
A method for fermenting Ganoderma lucidum supernatant and probiotics in a combined manner is similar to example 2, wherein the ganoderma lucidum submerged fermentation product obtained in example 2 is centrifuged at 5000r/min for 20min to obtain Ganoderma lucidum fermentation supernatant, and the Ganoderma lucidum fermentation supernatant is transferred to a fermenter, and then 50mL of supplementary culture medium (the culture medium contains 5g of fructo-oligosaccharide, 3g of glucose, 5g of rice peptide and 2g of folic acid) is added, and after PH adjustment, probiotics are inoculated for carrying out the combined fermentation.
Experimental example 1 evaluation experiment of growth and coupling conditions of thallus during fermentation
The plate count method was used to determine the time to reach the maximum viable count of the probiotic bacteria, the viable count of the probiotic bacteria, and the dry weight of the coupled fermentate cells in examples 1,2, and comparative examples 1,2, and 3, respectively. As is clear from Table 2, the bacterial suspensions of examples 1 and 2 have high viable count, short time to reach maximum viable count, and large dry weight of coupled ferment bacterial cells, which indicates that the invention is more beneficial to the growth of probiotics and the coupled fermentation of the probiotics and ganoderma lucidum than the comparative example. In addition, compared with comparative examples 2 and 3, the invention only needs to perform one sterilization operation on the fermentation tank and can perform coupling fermentation without transferring the ganoderma lucidum mycelia to the tank, and the coupling fermentation product can be obtained through one centrifugation and one batch of drying, thereby realizing simpler operation and better thallus growth and coupling effect under the condition of lower energy consumption.
TABLE 2 determination of probiotic bacterial growth in examples and comparative examples
| Sample name | Viable count CFU/mL | Time h for reaching maximum viable count | Dry weight g/L of fermented product thallus |
| Example 1 | 2.91×108 | 12 | 20.46 |
| Example 2 | 3.32×108 | 12 | 25.57 |
| Comparative example 1 | - | - | 18.84 |
| Comparative example 2 | 2.42×108 | 12 | 20.23 |
| Comparative example 3 | 2.09×108 | 12 | 20.02 |
Experimental example 2 effective ingredient detection experiment
(1) The polysaccharide components of the above five fermentation supernatants (examples 1, 2, comparative examples 1, 2 and 3) were analyzed. Slowly adding 4 times of absolute ethyl alcohol into the fermentation liquid, stirring uniformly, standing at 4 ℃ for alcohol precipitation for 14h, centrifuging at 8000r/min for 25min, collecting precipitate, washing the precipitate with ethanol of the same concentration for 3 times, adding distilled water into the precipitate to fully dissolve, heating and volatilizing to remove ethanol, freeze-drying to obtain extracellular crude polysaccharide, and measuring the polysaccharide content by a phenol sulfuric acid method. As can be seen from Table 3, the polysaccharide content of the coupled fermentation supernatants of examples 1 and 2 is 7.04g/L and 7.72g/L, respectively, while the polysaccharide content of the submerged fermentation supernatant of ganoderma lucidum of comparative example 1 is 2.27g/L, and it can be seen that the coupled fermentation of the invention increases the polysaccharide content, which not only increases the biological activities such as oxidation resistance and the like imparted by extracellular polysaccharide, but also reduces the waste of active ingredients in the fermentation supernatant of ganoderma lucidum mycelia, and the interaction is also beneficial to better utilization of the active ingredients by organisms. In addition, compared with the submerged fermentation supernatant of the ganoderma lucidum which is not subjected to coupling fermentation, the coupling fermentation supernatant also contains citric acid and lactic acid components through HPLC detection.
(2) The five above-mentioned fermentation supernatants (examples 1, 2, comparative examples 1, 2 and 3) were analyzed for ganoderma triterpenes. And (3) respectively adding 0.9mL of absolute ethyl alcohol into 0.1mL of the five sample solutions by using a vanillin-perchloric acid color development method, standing for 1h at room temperature, volatilizing the liquid by using a boiling water bath until the liquid is dried, taking a blank as a control group, adding 0.4mL of 5% vanillin-glacial acetic acid solution and 1mL of perchloric acid into the fermentation liquid and the blank group, rapidly and uniformly mixing, heating in a water bath at 65 ℃ for 15min, measuring the absorbance at a wavelength of 550nm, and calculating the triterpene content in the fermentation liquid by using a standard curve. As can be seen from Table 3, the Ganoderma triterpene content is the supernatant of the fermented product prepared in example 2, example 1, comparative example 2, comparative example 3 and comparative example 1 in order from the large to the small, which shows that the invention is beneficial to improving the Ganoderma triterpene content in the fermented product.
TABLE 3 comparison of the active ingredient analysis of examples and comparative examples
| Sample name | Polysaccharide content g/L | Ganoderma lucidum triterpene g/L |
| Experimental example 1 | 7.04 | 1.5 |
| Example 2 | 7.72 | 1.7 |
| Comparative example 1 | 2.27 | 0.8 |
| Comparative example 2 | 6.31 | 1.3 |
| Comparative example 3 | 5.35 | 1.1 |
Experimental example 3 antioxidation efficacy test experiment
(1) DPPH free radical scavenging ability
The DPPH radical scavenging ability of the fermentation supernatants was measured by the method described in example 4, taking 1mL each of the five above (examples 1, 2, comparative examples 1, 2 and 3).
(2) Super oxyanion (.O2 -) scavenging ability
The capacity of the fermentation supernatant to remove superoxide anions is determined by adopting a pyrogallol autoxidation method. 0.5mL of each of the five fermentation supernatants (examples 1,2, comparative examples 1,2 and 3) was taken in 10mL test tubes, 4.5mL of 0.05mol/LTris-HCI buffer solution and 4mL of distilled water were added, the mixture was shaken well and left to stand at room temperature for 10min, and then 0.3mL of 3mmol/L of pyrogallol solution was added, and the absorbance at 320nm was measured immediately after mixing. The values were recorded every 30s over 3min, the slope of the change was determined, the blank was replaced with the same volume of distilled water, and the clearance to .O2 - was calculated as follows:
Clearance (%) = (1-K i/K0) ×100% in the formula, K i is absorbance of the sample to be measured, and K 0 is absorbance of the blank control.
Table 4 comparison of antioxidant activity of examples and comparative examples
As can be seen from Table 4, the supernatant DPPH free radical scavenging rates of the fermented products prepared in test examples 1,2 and comparative examples 1,2 and 3 are 88.45%, 91.07%, 66.15%, 85.98% and 83.43%, respectively, and the O 2 - scavenging rates are 17.47%, 18.24%, 7.62%, 16.23% and 13.76%, respectively, and the antioxidant activities of examples 1 and 2 are higher than those of other methods, and the fermented products have good antioxidant effects.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.
Claims (10)
1. A coupled fermentation method of ganoderma lucidum and probiotics is characterized by comprising the following steps: (1) Activating, culturing, expanding culturing and fermenting the ganoderma lucidum to obtain a ganoderma lucidum submerged fermentation product; (2) Directly inoculating probiotics in the late stage of the submerged fermentation of the ganoderma lucidum, and carrying out coupling fermentation under proper conditions; (3) Freeze drying, vacuum drying under reduced pressure or spray drying the thallus layer and fermentation supernatant obtained by coupling fermentation, and obtaining Ganoderma and probiotic composite thallus dry powder, and Ganoderma and probiotic coupling fermentation supernatant dry powder for use .
2. The coupled fermentation method of ganoderma lucidum and probiotics according to claim 1, wherein the culture supernatant is directly inoculated with probiotics to ferment the probiotics in the fermentation tank of the late fermentation stage of ganoderma lucidum without centrifugal separation and tank transfer sterilization.
3. The method according to claim 1, wherein a supplementary nutrient medium is added to the submerged fermentation of ganoderma lucidum prior to inoculating probiotics for coupled fermentation.
4. The ganoderma lucidum and probiotic coupled fermentation method according to claim 3, wherein the optimal nutrient medium is supplemented with the following formula: adding 50mL of supplementary culture medium into every 1000mL of ganoderma lucidum submerged fermentation; every 50mL of the supplementary culture medium contains 5g of fructo-oligosaccharide, 3g of glucose, 5g of rice peptide and 2g of folic acid.
5. The ganoderma lucidum and probiotic coupled fermentation method according to claim 1, wherein the probiotic is lactic acid bacteria, bifidobacteria or a combination of both.
6. The method of claim 5, wherein the lactic acid bacteria in the probiotics are lactobacillus rhamnosus and the bifidobacteria in the probiotics are bifidobacterium longum.
7. The coupled fermentation method of ganoderma lucidum and probiotics according to claim 1, wherein the coupled fermentation culture device can perform aerobic fermentation and anaerobic fermentation simultaneously, and can adjust stirring speed and ventilation quantity to control dissolved oxygen quantity.
8. The ganoderma lucidum and probiotic coupled fermentation method according to claim 1, wherein the drying is freeze-drying, vacuum drying under reduced pressure or spray-drying.
9. A coupled fermentation product of ganoderma lucidum and probiotics, wherein the coupled fermentation product is prepared by the preparation method of any one of claims 1-8.
10. Use of the ganoderma lucidum and probiotic coupled fermentation method according to any one of claims 1-8 and the method for obtaining substances in cosmetics and health food.
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