Method for producing bioactive substances by utilizing actinomycetes to ferment fruit juice waste liquid

Abstract

The invention discloses a method for producing bioactive substances by utilizing actinomycetes to ferment fruit juice waste liquid, and belongs to the technical field of fermentation. According to the method, the fruit juice waste liquid is fermented and degraded by utilizing actinomycetes through the processes of screening, identifying, culturing, fermenting and extracting, so that organic substances can be degraded, bioactive substances with high additional value can be produced, and the secreted bioactive substances have broad-spectrum antibacterial activity; the compound has a remarkable inhibition effect on common pathogenic bacteria such as salmonella, escherichia coli and staphylococcus aureus in food, and has important economic and environment-friendly values.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes. Background Art

[0002] The juice industry produces a large amount of waste liquid when extracting juice, which mainly includes peels, cores, pulp residues and some unused liquid. If these by-products are discharged directly without treatment, it will not only waste resources, but also cause serious pollution to the environment.

[0003] With the global emphasis on sustainable development and environmental protection, wastewater treatment technology in the juice industry has also developed rapidly. At present, the treatment technologies for wastewater in the juice industry mainly include physical, chemical and biological methods. The physical method separates solid waste through filtration, centrifugation and other means to reduce the volume of wastewater; the chemical method uses chemical reactions to convert harmful substances in wastewater into harmless substances; and the biological method converts wastewater into valuable products such as biofuels and organic fertilizers through methods such as microbial fermentation. In addition, some emerging technologies, such as nanotechnology, membrane separation technology and supercritical fluid technology, also show great potential in the treatment of wastewater in juice. These technologies can more efficiently separate and recover useful components in wastewater, reduce environmental pollution, and improve resource utilization.

[0004] The treatment of waste liquid from the juice industry is not only related to environmental protection, but also an important way to improve the economic benefits of the industry. Through technological innovation and process improvement, the generation of waste liquid can be effectively reduced, the recycling of resources can be achieved, and the green development of the juice industry can be promoted. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for producing bioactive substances by fermenting fruit juice waste liquid with actinomycetes, so as to solve the technical problem that it is difficult to efficiently and economically treat fruit juice waste liquid.

[0006] To achieve the above object, the present invention provides a method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes, comprising the following steps:

[0007] Screening: Isolation of actinomycete strains from fruit juice wastewater;

[0008] Identification: Gene sequencing of the actinomycete strain is performed to determine its species;

[0009] Cultivation: selecting culture conditions according to the determined species to culture the actinomycete strain;

[0010] Fermentation: inoculating the cultured actinomycete strain into a fermentation matrix for fermentation to obtain a fermentation liquid, wherein the fermentation matrix includes the fruit juice waste liquid;

[0011] Extraction: centrifuging the fermentation broth and collecting the supernatant, filtering the supernatant to obtain the bioactive substance; or, mixing the collected supernatant with an organic solvent to obtain an organic phase, and concentrating the organic phase to obtain the bioactive substance.

[0012] In some embodiments of the present invention, in the screening step, the culture medium comprises a humic acid-vitamin selection medium.

[0013] In some embodiments of the present invention, in the culturing step, the culturing temperature is 27° C.-32° C.; and / or the culturing pH is 6.5-7.0; and / or the culturing time is 48 hours to 72 hours.

[0014] In some embodiments of the present invention, the fruit juice waste liquid and water are mixed in a mass ratio of 1:(3-4.5) to obtain the fermentation substrate.

[0015] In some embodiments of the present invention, in the fermentation step, the pH of the fermentation is 7.0 and / or the fermentation time is 48 hours to 72 hours.

[0016] In some embodiments of the present invention, in the fermentation step, the inoculation amount of the actinomycete strain is 2% of the total mass of the fermentation substrate.

[0017] In some embodiments of the present invention, in the fermentation step, the pH and residual sugar content of the fermentation substrate are measured every 12 hours, and the pH of the fermentation substrate is adjusted and / or glucose is supplemented according to the measurement results.

[0018] The present invention also provides a bioactive substance produced by the method for producing bioactive substances by fermenting fruit juice waste liquid with actinomycetes as described above.

[0019] The present invention also provides an application of the above-mentioned bioactive substance in the field of food and medicine, wherein the bioactive substance has an antibacterial effect.

[0020] In some embodiments of the present invention, the bioactive substance has an inhibitory effect on Escherichia coli, Staphylococcus aureus, and Salmonella.

[0021] The beneficial effects that can be achieved by the present invention are:

[0022] The invention utilizes fruit juice waste liquid to separate and screen to obtain a specific actinomycete strain, and cultivates the specific actinomycete strain. The cultured actinomycete strain is used to treat fruit juice waste liquid, which can not only degrade organic matter in the fruit juice waste liquid, but also produce bioactive substances with high added value. The secreted bioactive substances have broad-spectrum antibacterial activity, have a significant inhibitory effect on common pathogenic bacteria in food such as Salmonella, Escherichia coli and Staphylococcus aureus, and have important economic and environmental protection value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0024] Figure 1 The present invention is a flow chart of a method for producing bioactive substances by fermenting fruit juice waste liquid with actinomycetes according to an embodiment of the present invention.

[0025] Figure 2 Schematic diagram of the antibacterial effect of the bioactive substances extracted from Examples 1 to 3 of the present invention.

[0026] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0027] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0030] The present invention provides a method for producing biologically active substances by using actinomycete fermentation fruit juice waste liquid. The extraction method comprises the following steps:

[0031] Screening: Isolation of actinomycete strains from fruit juice wastewater;

[0032] Identification: Gene sequencing of the actinomycete strain is performed to determine its species;

[0033] Cultivation: selecting culture conditions according to the determined species to culture the actinomycete strain to obtain an actinomycete group;

[0034] Fermentation: inoculating the cultured actinomycete strain into a fermentation matrix for fermentation to obtain a fermentation liquid, wherein the fermentation matrix includes the fruit juice waste liquid;

[0035] Extraction: centrifuging the fermentation broth and collecting the supernatant, filtering the supernatant to obtain the bioactive substance; or, mixing the collected supernatant with an organic solvent to obtain an organic phase, and concentrating the organic phase to obtain the bioactive substance.

[0036] In the screening step, the present invention separates actinomycete strains from the fruit juice waste liquid, and then utilizes the separated actinomycete strains to ferment and degrade the fruit juice waste liquid, thereby being able to treat the fruit juice waste liquid in a targeted and efficient manner.

[0037] In the present invention, the fruit juice waste liquid includes waste generated in the production process of fruit juice beverages, and its production raw materials include fruits and / or vegetables. For example, in the production process of fruit juice beverages, the solid part remaining after filtration, as well as the waste water generated in the production process, and the waste fruit peels, vegetable residues, etc. generated by the pre-treatment of fruits and vegetables. The above waste materials usually contain a large amount of sugars and dietary cellulose, and may also contain at least one of proteins, oils, amino acids, taurine, caffeine, electrolytes, xylitol, mineral salts, and plant extracts. If discarded, it will cause waste. The present invention uses the above organic matter as a carbon source and utilizes a biological fermentation method to ferment and degrade the fruit juice waste liquid. It is efficient and economical, and can separate high-value-added biologically active substances. The secreted biologically active substances have broad-spectrum antibacterial activity and have a significant inhibitory effect on common pathogenic bacteria in food such as Salmonella, Escherichia coli and Staphylococcus aureus. The entire scheme has important economic and environmental value.

[0038] In the screening step, the actinomycete strains in the fruit juice waste can be screened out through the culture medium. Exemplarily, the fruit juice waste is dripped into the HV selection culture medium, and several actinomycete strains are obtained through culture.

[0039] In some embodiments, the components of the HV selection medium include: humic acid, CaCO3, Na2HPO4, MgSO4·7H2O, KCl, FeSO4·7H2O, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, inositol, biotin, p-aminobenzoic acid and agar.

[0040] In some embodiments, the contents of various components in the HV selection medium are as follows: 100 mg / L humic acid, 20 mg / L CaCO3, 500 mg / L Na2HPO4, 50 mg / L MgSO4·7H2O, 1700 mg / L KCl, 10 mg / L FeSO4·7H2O, 0.5 mg / L vitamin B1, 0.5 mg / L vitamin B2, 0.5 mg / L vitamin B3, 0.5 mg / L vitamin B5, 0.5 mg / L vitamin B6, 0.5 mg / L inositol, 0.25 mg / L biotin, 0.5 mg / L p-aminobenzoic acid and 18000 mg / L agar.

[0041] After completing the screening of actinomycete strains, it is necessary to identify the species of the strains and carry out targeted cultivation based on the species characteristics, which is conducive to obtaining more active and numerous actinomycete strains and achieving efficient degradation of fruit juice waste liquid.

[0042] In some embodiments, 16S rRNA gene sequencing is used to determine the identity of the actinomycete strain.

[0043] In some embodiments, the actinomycetes isolated from the fruit juice waste liquid belong to the genus Streptomyces.

[0044] In some embodiments, appropriate culture conditions are selected according to the species of the determined actinomycete strain for cultivation. For example, when the species of the actinomycete strain is determined to be Streptomyces, the culture temperature of the culture process is 37°C, the culture pH is 7.0, and the culture time is 48 hours to 72 hours. Under the above culture conditions, it is conducive to cultivating actinomycetes with strong activity and large quantity.

[0045] In the fermentation step, the fruit juice waste liquid is used as a part of the fermentation matrix, and the fermentation matrix is ​​fermented using the isolated and cultured actinomycetes, which can not only degrade the organic matter in the fruit juice waste liquid, but also produce bioactive substances with high added value. The secreted bioactive substances have broad-spectrum antibacterial activity and have a significant inhibitory effect on common pathogenic bacteria in food such as Salmonella and Escherichia coli, and have important economic and environmental value.

[0046] The fruit juice waste liquid is usually acidic, which may affect the fermentation and degradation of organic matter by actinomycetes. Therefore, in some embodiments, appropriate water can be added to the fermentation matrix to dilute the fruit juice waste liquid to obtain a suitable pH environment, such as a pH of 7.0. Exemplarily, the fruit juice waste liquid and water are mixed in a mass ratio of 1: (3-4.5) to obtain the fermentation matrix.

[0047] In some embodiments, the inoculation amount of the actinomycete strain is 1.5% to 3% of the total mass of the fermentation substrate, and can be any value within the range of 1.5%, 2%, 2.5%, 3%, etc.

[0048] In some embodiments, during the fermentation step, the pH and residual sugar content in the fermentation substrate are measured every 12 hours, and the pH of the fermentation substrate is adjusted and / or glucose is supplemented according to the measurement results.

[0049] When the pH is less than 6.3, the pH is adjusted to 7.0 to provide a suitable working environment for the fermentation and degradation of actinomycetes. In this embodiment, fermentation can be carried out in a fermenter, and the fermentation pH is set to 7.0. When the pH drops or rises, the peristaltic pump of the fermenter adjusts the pH to the set pH.

[0050] In some embodiments, the residual sugar content of the fermentation substrate may be measured every 12 hours. If the residual sugar content is found to be too low, glucose may be appropriately supplemented.

[0051] In some embodiments, glucose is supplemented during the fermentation process, the supplementation rate in the early stage of fermentation is 5ml / min to 7ml / min, and the supplementation rate in the later stage of fermentation is 10ml / min to 12ml / min. For example, glucose can be continuously supplemented at a rate of 5ml / min to 7ml / min before 24h of fermentation, and glucose can be continuously supplemented at a rate of 5ml / min to 7ml / min after 24h of fermentation. Timely supplementation of glucose can timely supplement the energy required for fermentation for actinomycetes, which is conducive to the complete degradation of fruit juice waste liquid and improves the fermentation degradation effect of fruit juice waste liquid.

[0052] In some embodiments, the glucose supplemented has a mass concentration of 60%.

[0053] In some embodiments, the fermentation temperature is 30° C., which can provide a suitable fermentation and degradation environment for the actinomycete strains and improve the degradation effect and efficiency of the fruit juice waste liquid.

[0054] In some embodiments, the fermentation time is 48 hours to 72 hours, which is conducive to the complete fermentation and degradation of organic matter in the fruit juice waste liquid and the production of more bioactive substances.

[0055] After the fruit juice waste liquid is fermented and degraded by the method of the present invention, a fermentation liquid is obtained. In addition to some large-volume impurities that have not been degraded, the fermentation liquid also contains a large amount of biologically active substances dissolved in the liquid. Therefore, in the extraction step, the fermentation liquid is centrifuged to collect the supernatant, so that the biologically active substances and the large-volume solid impurities can be separated, and the supernatant can be filtered to obtain the biologically active substances.

[0056] In some embodiments, the centrifugal speed for collecting the supernatant is 7000 r / min to 8000 r / min, the centrifugal time is 15 min to 20 min, and the centrifugal temperature is 2° C. to 4° C., which is conducive to obtaining a supernatant rich in bioactive substances.

[0057] In some embodiments, after the fermentation broth is centrifuged to obtain the supernatant, the supernatant and an organic solvent can be mixed to obtain an organic phase, the organic phase is collected, and the organic phase is concentrated to obtain the bioactive substance. The bioactive substance collected in this embodiment has high purity and good quality.

[0058] In some embodiments, the organic solvent comprises ethyl acetate.

[0059] In some embodiments, the volume ratio of the organic solvent to the supernatant is 1 to 3:1.

[0060] In some embodiments, after the organic solvent and the supernatant are mixed, the container is tightly covered and shaken to dissolve the bioactive substance in the supernatant into the organic phase.

[0061] In some embodiments, the organic phase is placed in a rotary evaporator, and the organic solvent is removed by rotary evaporation under reduced pressure to obtain concentrated bioactive substances.

[0062] The bioactive substance of the present invention has an antibacterial effect and can be applied to the field of food and medicine. For example, it has a good antibacterial effect on Escherichia coli, Salmonella and Staphylococcus aureus.

[0063] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0064] The following examples and comparative examples all use the same batch of juice waste liquid produced by preparing apple beverages as the treatment object.

[0065] Example 1

[0066] The method of producing biologically active substances by using actinomycete fermentation wastewater in this embodiment is as follows:

[0067] Screening: Actinomycete strains were isolated from the fruit juice waste liquid using HV selection medium. The composition of the HV selection medium is shown in Table 1.

[0068] Identification: The isolated actinomycete strain was sequenced by 16S rRNA gene, and the results showed that the actinomycete strain belonged to the genus Streptomyces.

[0069] Cultivation: The isolated actinomycete strains were cultured at 30°C and pH 7.0.

[0070] Fermentation: The fruit juice waste liquid and water are mixed in a mass ratio of 1:4 to prepare a fermentation matrix, the pH of the fermentation matrix is ​​adjusted to 7.0, and the cultured actinomycete strain is inoculated into the fermentation matrix for fermentation for 72 hours. Based on the total volume L of the fermentation matrix, the inoculation amount of the actinomycete strain is 2%. In this step, the pH and residual sugar content in the fermentation matrix are measured every 12 hours, and the pH is adjusted to 7.0 according to the measurement results, and glucose is supplemented.

[0071] Extraction: The fermentation broth was centrifuged and the supernatant was collected, and the supernatant was filtered to obtain the bioactive substances.

[0072] Example 2

[0073] Example 2 The fruit juice waste liquid was fermented and degraded with reference to Example 1, and the bioactive substances were separated, except that the fruit juice waste liquid and water were mixed in a mass ratio of 1:4.5 to obtain the fermentation matrix.

[0074] Example 3

[0075] Example 3 refers to Example 1 to ferment and degrade the fruit juice waste liquid, and separate the bioactive substances. The difference is that the fruit juice waste liquid and water are mixed in a mass ratio of 1:3.5 to obtain the fermentation matrix.

[0076] Table 1

[0077]

[0078]

[0079] Performance Testing

[0080] The method for treating fruit juice waste liquid of the present invention can extract biologically active substances, and the extracted biologically active substances have an inhibitory effect on various pathogens such as Escherichia coli, Staphylococcus aureus and Salmonella. Therefore, the antibacterial test of the biologically active substances extracted from Examples 1 to 3 was carried out by using a paper diffusion method, and the operation is as follows:

[0081] Use sterile cotton swabs to take appropriate amounts of Escherichia coli suspension, Staphylococcus aureus suspension and Salmonella suspension (the turbidity is 0.5), and spread the bacterial suspension evenly on the surface of the culture dish, applying it once horizontally and vertically to ensure that the entire culture medium surface is covered with bacteria.

[0082] A sterilized blank filter paper (0.7 mm) from Beekman Bio was immersed in the extracted bioactive substances for 10 s, and then the filter paper was gently placed on the surface of the culture dish inoculated with microorganisms.

[0083] The culture dish was placed upside down in a constant temperature incubator at 37°C for 24 hours.

[0084] After the culture is completed, take out the culture dish, observe the antibacterial zone around the filter paper, use a ruler to measure the diameter of the clear antibacterial zone around the filter paper, and record the result in mm. Figure 2 and Table 2, where Figure 2 A represents the sterile water control group, B represents Example 1, C represents Example 2, and D represents Example 3.

[0085] Table 2

[0086]

[0087]

[0088] From Table 2 and Figure 2It can be known that the method for treating fruit juice waste liquid of the present invention can not only degrade organic matter in the fruit juice waste liquid, but also produce bioactive substances with high added value. The secreted bioactive substances have broad-spectrum antibacterial activity and have significant inhibitory effects on common pathogenic bacteria in food such as Salmonella, Escherichia coli and Staphylococcus aureus, and the antibacterial effects on Escherichia coli and Staphylococcus aureus are particularly significant. The method for treating fruit juice waste liquid of the present invention has important economic and environmental protection values.

[0089] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes, characterized in that: The following steps are involved: Screening: Isolation of actinomycete strains from fruit juice wastewater; Identification: Gene sequencing of the actinomycete strain is performed to determine its species; Cultivation: selecting culture conditions according to the determined species to culture the actinomycete strain; Fermentation: inoculating the cultured actinomycete strain into a fermentation matrix for fermentation to obtain a fermentation liquid, wherein the fermentation matrix includes the fruit juice waste liquid; Extraction: centrifuging the fermentation broth and collecting the supernatant, filtering the supernatant to obtain the bioactive substance; Alternatively, the collected supernatant is mixed with an organic solvent to obtain an organic phase, and the organic phase is concentrated to obtain the bioactive substance.

2. The method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes according to claim 1, characterized in that: In the screening step, the culture medium comprises a humic acid-vitamin selection medium.

3. The method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes according to claim 1, characterized in that: In the culturing step, the culturing temperature is 27° C.-32° C.; and / or the culturing pH is 6.5-7.0; and / or the culturing time is 48 hours to 72 hours.

4. The method for producing biologically active substances by using actinomycete fermentation wastewater according to any one of claims 1 to 3, characterized in that: The fruit juice waste liquid and water are mixed in a mass ratio of 1:(3-4.5) to obtain the fermentation substrate.

5. The method for producing biologically active substances by using actinomycete fermentation wastewater according to any one of claims 1 to 3, characterized in that: In the fermentation step, the pH of the fermentation is 6.5-7.0 and / or the fermentation time is 48 hours to 72 hours.

6. The method for producing biologically active substances by using actinomycete fermentation wastewater according to any one of claims 1 to 3, characterized in that: In the fermentation step, the inoculation amount of the actinomycete strain is 2% of the total mass of the fermentation substrate.

7. The method for producing biologically active substances by fermenting fruit juice waste liquid with actinomycetes according to any one of claims 1 to 3, characterized in that: In the fermentation step, the pH of the fermentation substrate is measured once every 12 hours, and the pH of the fermentation substrate is adjusted according to the measurement result; and / or the residual sugar content of the fermentation substrate is measured once every 12 hours, and glucose is supplemented according to the measurement result.

8. A bioactive substance produced by the method for producing bioactive substances by fermenting fruit juice waste liquid with actinomycetes as claimed in any one of claims 1 to 7.

9. Use of the bioactive substance according to claim 8 in the field of food and medicine, characterized in that: The bioactive substance has an antibacterial effect.

10. The use according to claim 9, characterized in that: The biologically active substance has an inhibitory effect on Escherichia coli, Staphylococcus aureus and Salmonella.

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