Citrus processing waste dehydration method and application of dehydrated liquid

Abstract

Aiming at series feed utilization problems caused by high moisture content of processing wastes of fruits and vegetables such as oranges and the like, the invention provides the dehydrating agent, the dehydrating agent comprises main components and secondary components, the main components comprise citric acid, sodium bicarbonate, sodium carbonate and quick lime, and the secondary components comprise one or more of pectinase, cellulase and xylanase; wherein the composition proportion of the main components is 1: (1-5), the composition proportion of the secondary components is 1: (1-5), and the proportion of the main components to the secondary components is 1: (0.02-0.5). Meanwhile, the invention also provides a low-cost physicochemical dehydration method and a resource utilization method of the dehydrated liquid. The method not only solves the problems that the processing wastes of fruits and vegetables such as citrus are easy to decay, high in transportation cost and difficult to crush and process, but also turns wastes into wealth, and the eluate is applied to culture of microorganisms and production and accumulation of high-value metabolites of the microorganisms, so that the comprehensive resource utilization of the processing wastes of citrus is realized; the method is helpful for realizing'grain storage in technology 'and'comprehensive and multi-way food resource development'.

Description

Technical Field

[0001] This invention relates to the reuse of biological resources, and particularly to the disposal and reuse of citrus processing waste. Technical Background

[0002] Over the past decade, my country's citrus production has increased from 36.1753 million tons to 67.9149 million tons, with production continuously rising. Approximately 30% of the citrus fruit is used for deep processing annually, generating a large amount of citrus processing waste. Due to the concentrated ripening and processing time of citrus fruits, the processing waste is highly seasonal, has a high moisture content, is easily perishable, and is inconvenient to transport. It is often discarded by processing plants nearby or landfilled as agricultural waste, resulting not only in resource waste but also soil pollution and ecological imbalance. Citrus processing waste is high in soluble sugars and pectin, which microorganisms can use as a carbon source for growth and reproduction, as well as the production and accumulation of beneficial products such as enzymes and proteins. This waste can be developed into unconventional feed ingredients, reducing the proportion of bulk feed ingredients, ensuring a balance between feed supply and demand, and alleviating ecological pressure.

[0003] The primary challenge in utilizing citrus processing waste for animal feed is addressing the difficulties in storage, processing, and transportation caused by its high moisture content. Currently, the main methods for reducing the moisture content of high-moisture raw materials are mechanical pressing and heat drying. However, citrus processing waste is already a product of mechanical pressing and is difficult to dehydrate further through mechanical pressure. Furthermore, the sugars and pectin in citrus processing waste cause plant tissue cells to clump together, making it prone to clumping. Drying this waste not only incurs high energy costs but also easily results in a situation where the outside is scorched while the inside remains damp. Summary of the Invention

[0004] This invention addresses the challenges of utilizing high moisture content in the processing waste of citrus fruits and vegetables for animal feed, providing a low-cost physicochemical dehydration method and a method for resource utilization of the dehydrated liquid. The proposed physicochemical dehydration method aims to solve the problems of easy spoilage, high transportation costs, and difficulty in crushing and processing citrus and other fruit and vegetable processing waste.

[0005] The objective of this invention is achieved through the following measures: A dehydrating agent comprising a main component and a minor component, wherein the main component is citric acid, sodium bicarbonate, sodium carbonate and quicklime, and the minor component is one or more of pectinase, cellulase and xylanase; wherein the composition ratio of the main component is 1:1 to 5, the composition ratio of the minor component is 1:1 to 5, and the ratio of the main component to the minor component is 1:0.02 to 0.5.

[0006] The above-mentioned dehydrating agent is used in the treatment of processing waste from citrus and other fruits and vegetables. The method of using the dehydrating agent includes: when the citrus processing waste contains a large amount of citrus peel and the tubers are ≥3cm, they need to be crushed to ≤1cm; mix 0.6~0.86% (w / w) of the dehydrating agent evenly with the citrus processing waste, allow it to stand at a temperature below 40℃ for 18~24h, and then centrifuge at 2000r / min for 5~10min to separate the solid and liquid. The liquid is the extract, and the solid is the dehydrated citrus processing waste.

[0007] Preferably, the container used for the static reaction of citrus processing waste and dehydrating reagent is a container with a smooth inner wall and a liquid outlet to facilitate the discharge and collection of the dehydrated liquid during the reaction. After the dehydration reaction, centrifugation can be performed using a filter centrifuge or a sedimentation centrifuge. When the tuber particle size is 2mm < 1cm, a filter centrifuge with a filter cloth mesh size ≥ 80 mesh can be used for solid-liquid separation. When the tuber particle size is ≤ 2mm or the reacted material is further crushed to a particle size ≤ 2mm, a sedimentation centrifuge can be used for solid-liquid separation.

[0008] Citrus processing waste refers to the waste generated after the deep processing of citrus fruits, such as waste from the production of juice, canned goods, and citrus essential oils. It generally consists of substandard citrus fruits, citrus seeds, citrus pulp, citrus peels, and citrus segments. Due to different deep processing techniques, citrus processing waste can be roughly divided into three categories: Category 1 is high-moisture solid waste generated during citrus food processing; Category 2 is waste generated from the extraction of citrus essential oils from citrus peels; and Category 3 is a mixture of Categories 1 and 2.

[0009] Another objective of this invention is to provide a method for the resource utilization of the above-mentioned citrus processing waste effluent. This method aims to improve the downstream processes of citrus processing waste dehydration treatment, solve the problems of effluent treatment and discharge, and achieve comprehensive resource utilization of citrus processing waste.

[0010] The above-mentioned citrus processing waste effluent is used for microbial cultivation. A microbial culture medium includes the above-mentioned effluent. This biological culture medium can be used for strain propagation and the production and accumulation of organic acids, enzymes, and proteins.

[0011] The culture medium can be cultured for microorganisms including, but not limited to, lactic acid bacteria, Bacillus, and yeast; the genera of the culturable microorganisms include, but are not limited to, Lactobacillus plantarum, Bacillus subtilis, Bacillus belesiensis, Bacillus pumilus, Candida tropicalis, Saccharomyces cerevisiae, and Caribocchio Mayer's yeast.

[0012] Preferably, the culture medium further includes 10 g / L peptone, 0-10.5 g / L ammonium sulfate, and sodium hydroxide to adjust the pH to 5.5-7.4. It utilizes the citrus processing waste effluent as a carbon source, peptone and ammonium sulfate as supplementary nitrogen sources, and sodium hydroxide solution to adjust the pH to prepare the effluent microbial culture medium. Specifically: A lactic acid bacteria culture medium comprises: 500 mL / L of effluent, 10 g / L of peptone, 1.0~3.0 g / L of ammonium sulfate, 0.2 g / L of magnesium sulfate, 0.04 g / L of manganese sulfate, and pH adjusted to 5.5~5.9.

[0013] A Bacillus culture medium comprises: 500 mL / L of exudate, 10 g / L of peptone, 0-2.0 g / L of ammonium sulfate, 5.0 g / L of sodium chloride, and pH adjusted to 7.0-7.4.

[0014] A yeast culture medium, comprising 500 mL / L of extract, 10 g / L of peptone, 8.5-10.5 g / L of ammonium sulfate, and pH adjusted to 6.3-6.7.

[0015] The above culture medium was sterilized at 121℃ for 15 min, cooled to room temperature, and then inoculated with 2% of the secondary seed culture of the strain according to the volume of the culture medium. Lactic acid bacteria were cultured statically at 37℃, Bacillus was cultured at 37℃ and 220 r / min, and yeast was cultured at 30℃ and 220 r / min.

[0016] The method provided by this invention can effectively improve the comprehensive utilization of waste from the processing of citrus fruits and vegetables, alleviate ecological pressure, and help realize "food storage through technology" and "comprehensive and multi-faceted development of food resources". Beneficial effects

[0017] 1. The present invention provides a low-cost physicochemical dehydration method that releases bound water and intracellular water from citrus processing waste, followed by solid-liquid separation via centrifugation. The treated citrus processing waste is as follows: 1) The moisture content is reduced to 66%~77%, which is suitable for direct biological fermentation. 2) The color is light yellow, no different from before dehydration. The density of solids increases, making them easier to crush and process, which solves one of the difficulties in using citrus processing waste as feed. 3) Biomass weight is reduced by 30% to 45%, allowing for local processing at citrus processing plants, effectively reducing transportation costs and facilitating transportation; 4) Extended storage time: no obvious spoilage after 30 days at ≤37℃; 5) The pectin content is reduced by about 13%, and the content of major anti-nutritional factors is also reduced, which helps to improve the feed utilization efficiency of citrus processing waste.

[0018] 6) The dehydration treatment has a low cost, and the reagents used are safe, non-toxic, and require a small amount. The reagent usage is 6kg to 8.6kg per ton of citrus processing waste, with a reagent cost of approximately 27 to 128 yuan. Preferably, the reagent usage is 8kg per ton of citrus processing waste, with a reagent cost of approximately 33 yuan.

[0019] In addition, the dewatering liquid has a pH of 4.60~5.85, a yellowish color, and no odor, making it easy to utilize as a resource. The dewatering method provided by this invention can also be used in conjunction with mechanical pressing to improve the dewatering efficiency of mechanical pressing and the clarity of the dewatering liquid.

[0020] The present invention provides a method for the resource utilization of citrus processing waste effluent, aiming to solve the problem of large amounts of effluent generated during the reduction of moisture content in citrus processing waste. This effluent contains a large amount of sugars and a small amount of nitrogen; direct disposal would not only cause environmental pollution but also waste resources. Therefore, this invention proposes a method for the resource utilization of citrus processing waste effluent. While enabling the application of citrus processing waste as an unconventional feed ingredient, the effluent treated by this method can also be used for microbial cultivation and the generation and accumulation of high-value metabolites, reducing industrial fermentation costs and achieving the resource utilization of citrus processing waste. The resource utilization methods of citrus processing waste effluent provided by this invention mainly include the following two aspects: As an energy source, it provides energy for the growth and reproduction of various microorganisms, reducing the cost of microbial culture. After 5 days of culture, the viable cell counts of *Bacillus belyssioides* DP-2, *Bacillus subtilis* B92, *Candida tropicalis* GB3, and *Saccharomyces cerevisiae* RA2 were all higher than those in commercial culture media, with *Bacillus subtilis* B92 reaching a viable cell count of 1.03 × 10⁻⁶. 9 Although the viable count of Bacillus pumilus 2Y6 was lower than that of commercial culture medium, it still reached 1.2 × 10⁻⁶ CFU / mL. 7 CFU / mL.

[0021] As a basic component of lactic acid bacteria culture medium, it is used to produce and accumulate lactic acid. After 5 days of culture, the pH of Lactobacillus plantarum 28-7 and Lactobacillus plantarum 29-3 culture medium both dropped below 4.0, and the lactic acid contents were 19.84 and 20.95 mg / mL, respectively.

[0022] As an inducing substrate for Bacillus-secreted enzymes, it was used to induce Bacillus to secrete and accumulate enzymes. After 5 days of culture, the pectinase activities in Bacillus belyss DP-2, Bacillus subtilis B92, and Bacillus pumilus 2Y6 reached 1075.80 U / mL, 676.32 U / mL, and 1932.58 U / mL, respectively, which were much higher than those in commercial culture media; the cellulase activities reached 8.74 U / mL, 14.29 U / mL, and 19.42 U / mL, respectively, while cellulase activity was not detected in commercial culture media.

[0023] As a solvent for both liquid substrates and solid nitrogen sources, it was used to cultivate yeast and accumulate biological proteins. After 5 days of cultivation, the protein yields in the cultures of *Candida tropicalis* GB3, *Saccharomyces cerevisiae* RA2, and *Callibocae Mayerii* 2120 reached 6.94 g / L, 5.23 g / L, and 4.48 g / L, respectively. Among them, the protein content in the culture medium extracted from *Saccharomyces cerevisiae* RA2 was approximately 95.15% higher than that of commercially available cultures.

[0024] In summary, the method for resource utilization of citrus processing waste liquid provided by this invention is beneficial for transforming low-value waste into high-value products. Attached Figure Description

[0025] Figure 1 Citrus processing and the generation of processing waste.

[0026] Figure 2 A comparison of the colors of the effluent from different experimental groups in an orthogonal experiment.

[0027] Figure 3 Citrus processing waste liquid.

[0028] Figure 4 Effects of different dehydrating agents on the composition of citrus processing waste after dehydration: Note: 1) Figure 4 In A~4D, the grouped bar charts marked with * indicate significant differences; 2) Figure 4 In E, grouped bar charts labeled with different lowercase letters indicate significant differences.

[0029] Figure 5 Effects of different dehydrating agents on the composition of citrus processing waste liquid: Grouped bar charts with different lowercase letters indicate significant differences.

[0030] Figure 6 Optimization of physical and chemical dehydration parameters for citrus processing waste: Grouped bar charts with different lowercase letter labels indicate significant differences.

[0031] Figure 7 Classification of citrus processing waste.

[0032] Figure 8 The effects of physicochemical dehydration methods on the moisture content of different types of citrus processing waste and the pH of the dehydrated liquid.

[0033] Figure 9 Galacturonic acid standard curve was used to determine pectinase activity.

[0034] Figure 10 Reducing sugar standard curve for cellulase activity assay.

[0035] Figure 11The dehydration process of citrus processing waste and the resulting dehydrated citrus processing waste and dewatering liquid. Detailed Implementation

[0036] The following experimental examples are further illustrative of the present invention and are not intended to limit the invention. Specific experimental conditions and methods are not specified in the following examples; conventional methods and conditions, or those selected according to the product instructions, are followed. "h" represents hours, "min" represents minutes, and r / min represents rotational speed per minute.

[0037] In this invention, all the materials and reagents described are commonly used in the field and can be obtained through conventional commercial channels.

[0038] Example 1

[0039] 1.1 Test Methods The citrus processing waste used in this case study came from a citrus juice processing plant in Zhongxian County, Chongqing. This plant extracts and recycles juice from citrus fruits, and the remaining solid waste is discharged from the waste outlet into transport vehicles, which then transport it back to this laboratory. The citrus processing waste used in this case study mainly includes substandard citrus fruits, citrus seeds, citrus pulp, citrus peel, and citrus segments, with a moisture content of approximately 85%. Figure 1 As shown.

[0040] Accurately weigh 1.00 kg of citrus processing waste, add reagents according to Table 1, mix well, and place at room temperature (mid-March in Chongqing) for 24 hours. Then, centrifuge using a filter centrifuge with a 16-mesh filter at 2000 rpm / min for 5 minutes. Record the weight of the dehydrated citrus processing waste, collect the leachate, and determine the pH of the leachate. Using the moisture content of the dehydrated citrus processing waste and the pH of the leachate as comprehensive indicators, the optimal reagent combination is screened. The moisture content after dehydration is calculated as follows: Moisture content (%) of citrus processing waste after dehydration = 100 - (dry matter content of citrus processing waste * weight of citrus processing waste before dehydration) / weight of citrus processing waste after dehydration × 100%.

[0041]

[0042] 1.2. Test Results Range (R) analysis showed that the order of influence on the moisture content of citrus processing waste was: sodium bicarbonate (B) > xylanase (G) > quicklime (D) > citric acid (A) > pectinase (F) > dextranase (H) > sodium carbonate (C) > cellulase (E). In the orthogonal experiment, the color of the effluent varied among different experimental groups, changing from light yellow to brownish-yellow with increasing pH. Figure 2As shown. Using dehydration rate and pH of the dehydrated liquid as comprehensive indicators, the optimal dehydrating agent composition was determined to be: citric acid, sodium bicarbonate, sodium carbonate, quicklime, cellulase, pectinase, and xylanase, with a ratio of 1:1:1:1:0.1:0.1:0.1. The amounts of citric acid, sodium bicarbonate, sodium carbonate, and quicklime were 0.2% (w / w) of the fresh weight of the citrus processing waste, and the amounts of cellulase, pectinase, and xylanase were 0.02% (w / w) of the fresh weight of the citrus processing waste. After dehydration, the color of the citrus processing waste did not change significantly, and the moisture content decreased from 84.78% to approximately 68%. The solid waste volume decreased, density increased, and viscosity decreased after dehydration, making it easier to pulverize. The dehydrated liquid was light yellow (…). Figure 3 It has a pH of around 5.7 and no odor.

[0043] Effects of different dehydrating agents on the composition of citrus processing waste and dewatering liquid after dehydration 2.1 Test Methods The citrus processing waste used in this case study came from a citrus juice processing plant in Zhongxian County, Chongqing. This plant extracts and recycles citrus juice, and the remaining solids are processed by scraping and peeling in a pulverizer. The larger, more structurally sound citrus peels are picked and packaged by workers for citrus essential oil extraction. The remaining pulp, seeds, smaller pieces, or broken peels are transported through the waste inlet to a truck and then back to our laboratory. Compared to the citrus processing waste used in Case Study 1, "Screening of Dehydrating Agent Components for Citrus Processing Waste," the citrus processing waste used in this case study has a higher viscosity, softer texture, and higher moisture content, approximately 86%.

[0044] Using no reagents as a control, and considering dehydration rate and reagent cost, this study investigated the effects of four different dehydrating agents and their influence on the composition of citrus processing waste and exudate after dehydration. The experiment consisted of five groups: Group 1, with a dehydrating agent composition of citric acid, sodium carbonate, sodium bicarbonate, quicklime, xylanase, pectinase, and cellulase in a ratio of 1:1:1:1:0.1:0.1:0.1; Group 2, with a dehydrating agent composition of citric acid, sodium carbonate, sodium bicarbonate, quicklime, and pectinase in a ratio of 1:1:1:1:0.1; Group 3, with a dehydrating agent composition of citric acid, sodium carbonate, sodium bicarbonate, and quicklime in a ratio of 1:1:1:1; Group 4, with a dehydrating agent composition of citric acid, sodium bicarbonate, and quicklime in a ratio of 1:1:1; and Group 5, a blank control group, using no reagents. Each group had three replicates, with 1.000 kg of citrus processing waste accurately collected for each replicate. The citrus processing waste was mixed thoroughly with the dehydrating reagent and allowed to stand at room temperature (mid-May in Chongqing) for 24 hours. Then, it was centrifuged at 2000 r / min for 5 min using a filter centrifuge with a 16-mesh filter. The extract was collected, and the moisture content of the dehydrated citrus processing waste and the weight of the extract / citrus processing waste were calculated. After dehydration, the citrus processing waste was dried at 65℃ until basically dry, then pulverized and sampled. The moisture, crude protein, soluble sugar, soluble protein, crude fiber, acid detergent fiber, neutral detergent fiber, lignin, and pectin contents of the sample were determined. Simultaneously, the pH, soluble sugar, acid-soluble protein, and total nitrogen contents of the extract were determined.

[0045] 2.2 Test Results The effects of different dehydrating agents on the composition content of citrus processing waste after dehydration, such as Figure 4 As shown. By Figure 4 As shown in A~4D, compared with the control group, the soluble sugar and moisture content of the dehydrated citrus processing waste in groups 1, 2, 3, and 4 were significantly reduced, while the ratio of dehydrated liquid weight to citrus processing waste weight was significantly increased. However, the soluble protein content in the citrus processing waste treated in groups 3 and 4 was significantly reduced. Figure 4 In group C and 4D, the amount of neutral detergent fiber in citrus processing waste was significantly increased in group 3 treatment. Figure 4 C). In addition, by Figure 4 E indicates that, compared with the control group, the pectin content in citrus processing waste treated in groups 1, 2, and 3 was significantly reduced. The effects of different dehydrating agent compositions on the exudate are as follows: Figure 5 As shown. Compared with the control group, the soluble sugar content in the exudate of different groups ( Figure 5 A) and acid-soluble proteins ( Figure 5 B) Content: There was no significant difference in content. Figure 5As shown in C, the nitrogen content in the effluent from groups 2 was significantly higher than that from the control group, while other groups showed no significant difference from the control group. Figure 5 As shown in D, the pH of the effluent from groups 1 to 3 was significantly higher than that of the control group, at 5.35, 5.43, and 5.41, respectively. The pH of the effluent from group 4 and the control group was 4.67 and 4.13, respectively. Overall, the pH of the effluent obtained from different groups was below 6.00.

[0046] In summary, from group 1 to group 4, the moisture content of the dehydrated citrus processing waste decreased sequentially, while the reagent dosage (6 kg~8.6 kg / ton) and reagent cost increased sequentially (27 yuan~128 yuan / ton). It is worth noting that the higher the pectin content, the greater the stickiness of the dehydrated citrus processing waste and the more difficult it is to pulverize. Among the four dehydrating agents, the pectin content in the citrus processing waste treated in groups 1-3 was significantly lower than that in the control group. Therefore, considering reagent dosage, reagent cost, dehydration effect, and pectin content in the dehydrated citrus processing waste, the preferred dehydrating agent composition is found in group 3, with a reagent dosage of 6 kg / ton and a reagent cost of 33 yuan / ton.

[0047] 3. Optimization of dehydration conditions for citrus processing waste 3.1 Test Methods The citrus processing waste used in this case study is consistent with that in Case Study 2, "Effects of Different Dehydrating Agents on the Composition of Dehydrated Citrus Processing Waste and Extract." The reaction time, reaction temperature, and centrifugation time of the reagents were optimized. The experimental factors and their levels are shown in Table 2. Each treatment was repeated in triplicate, with 1.000 kg of citrus processing waste accurately collected for each replicate. The citrus processing waste was thoroughly mixed with the dehydrating agent. The basic dehydration conditions were: citrus processing waste reacted with the dehydrating agent for 24 hours at room temperature (June in Chongqing). Then, a filter centrifuge with a 16-mesh filter was used, and the mixture was centrifuged at 2000 rpm for 5 minutes. The extract was collected, and the moisture content of the dehydrated citrus processing waste and the ratio of extract weight to citrus processing waste weight were calculated.

[0048]

[0049] 3.2 Test Results like Figure 6 As shown, the moisture content of citrus processing waste was lowest at 24 hours, significantly lower than at 6 hours and 12 hours, and not significantly different from that at 18 hours. Figure 6 A). Within the range of room temperature to 50°C, the reaction temperature has a significant impact on the moisture content and waste liquid weight / citrus processing waste weight in the dehydrated citrus processing waste. Figure 6(B) However, citrus processing waste will severely rot and deteriorate under conditions of 40℃ and 50℃. Regarding centrifugation time, the moisture content after centrifugation for 5 min and 10 min is significantly lower than that after 1 min and 3 min, and also lower than that after 4 min. In summary, considering economic cost and dehydration effect as comprehensive indicators, the preferred physicochemical dehydration parameters for citrus processing waste are: after the dehydration reagent is mixed evenly with the citrus processing waste, react at room temperature (<40℃) for 18 h~24 h, and then centrifuged at 2000 r / min for 5 min.

[0050] 4. The effects of physicochemical dehydration on processing waste from different types of citrus fruits 4.1 Test Methods like Figure 7 As shown, the citrus processing waste used in this case consists of three categories, all from a citrus juice processing plant in China Citrus City (Zhongxian County, Chongqing). Category 1 ((7A)) is the residue after citrus fruits have been cut, juiced, and the juice recycled. It enters the waste inlet via a conveyor belt and then enters a transport vehicle. It mainly consists of substandard citrus fruits, citrus peels, and citrus pulp, with a moisture content of about 75%, low viscosity, hard texture, and easy crushing. Category 2 (7B) is based on Category 1. Between the residue conveyor belt and the waste inlet, the equipment crushes and separates the peels and pulps. Workers then pick out and collect the larger, intact citrus peels for use in the extraction of citrus essential oils. The remaining part is Category 2, mainly consisting of citrus pulp and smaller or crushed citrus peels. This category has a high moisture content of 86.22%, a soft texture, higher viscosity, and stronger water retention capacity, making it difficult to crush and process. Category 3 (7C) is a mixture of Category 1 and Category 2. It occurs when there is a large volume of citrus processing and when the citrus peel is not sorted and collected in a timely manner. The texture, viscosity and water retention capacity of this category are between those of Category 1 and Category 2, and it has the largest output.

[0051] The dehydrating agent used in this experiment and its composition ratio are the same as those in Scheme 3 in the previous examples. Before conducting the dehydration experiment, the tubers ≥3cm in the processing waste of Class 1 and Class 3 citrus were coarsely crushed to ≤1cm, while the peel of Class 2 citrus was not crushed. The dehydrating agent for the citrus processing waste was mixed evenly, and the dehydration experiment was conducted under the optimal conditions in the previous examples.

[0052] 4.2 Experimental Results The same dehydration method resulted in inconsistent dehydration efficiency for different types of citrus processing waste, which may be related to the different types of waste. For example, Type 1 citrus processing waste has a lower moisture content and lower viscosity, while Types 2 and 3 citrus processing waste have higher viscosity, especially Type 2, which has a softer texture, higher moisture content, and stronger water retention capacity, resulting in a lower dehydration rate. As shown in Table 3, the moisture content of Types 1, 2, and 3 citrus processing waste decreased from 75.86%, 86.22%, and 84.78% to 68.44%, 77.18%, and 71.50%, respectively. Types 1 and 3 waste can be bio-fermented after dehydration without the addition of auxiliary materials. Type 2 waste can be dehydrated and crushed together with Type 1 waste, or fermented after dehydration with 10% (w / w) wheat bran. Furthermore, the pH of the effluent from all three types of waste was <6.00, odorless, and golden in color, making them suitable for short-term storage and reuse.

[0053] Example 2

[0054] 1. Detection Method Viable cell count determination in culture medium: 1 mL of the well-mixed bacterial culture medium was placed in a 9 mL sterile saline tube. Using a serial dilution method, 0.1 mL of a 10⁶ dilution was spread onto the culture medium of each suitable strain. The tubes were incubated upside down at 30℃~37℃ for 24 h, and colony counting was performed. In this example, the viable cell count range was ≥1.0×10⁷ CFU / mL.

[0055] Determination of lactic acid content in culture medium: The determination was made in accordance with the National Food Safety Standard for Food Additives Lactic Acid (GB1886.173—2016).

[0056] Determination of pectinase activity in culture medium: Take 1 mL of the bacterial culture medium, centrifuge at 10000 r / min for 2 min at 4℃, and dilute the supernatant appropriately to obtain the crude enzyme solution. Add 80 µL of pectin substrate to a 2 mL round-bottom centrifuge tube and preheat in a 55℃ water bath for 5 min. Add 80 µL of appropriately diluted crude enzyme solution to the experimental group, and no enzyme solution is added to the enzyme inactivation control group. Incubate in a 55℃ water bath for 30 min. After removal, add 200 µL of DNS reagent to each test tube. Then add 80 µL of inactivated crude enzyme solution (boiling water bath for 10 min) to the enzyme inactivation control group, mix well, boil in a boiling water bath for 10 min, immediately remove and cool under running water, add 625 µL of distilled water, mix well, and add 200 µL of the solution to a 96-well plate. Measure the absorbance at 540 nm using a microplate reader. The pectinase activity was calculated using the measured UV absorbance values ​​and substituted into the formula. Enzyme activity per unit volume (U / mL) = [(A1-A2)×1000×N] / (V×k×t), where A1 is the OD540 nm value of the experimental group, A2 is the OD540 nm value of the control group, 1000 represents 1 mg = 1000 μg, N is the dilution factor of the supernatant, V is the volume of diluted enzyme solution used for enzyme activity determination (mL), k is the slope of the galacturonic acid standard curve, and t is the reaction time (min). An enzyme activity unit is defined as: at 55℃ and pH 5.0, the production of 1 μg of galacturonic acid per minute from pectin hydrolysis is defined as one enzyme activity unit, denoted by U. The galacturonic acid standard curve needs to be redrawn each time the DNS solution is changed. The galactose standard curve used in this case is as follows: Figure 9 As shown.

[0057] Determination of cellulase activity in the culture medium: The determination was performed according to the standard "Determination of Cellulase Activity in Feed Additives - Spectrophotometry" (NY / T 912-2020). Cellulase activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 7.5 mg / mL sodium carboxymethyl cellulose solution at 37℃ and pH 5.5. One unit of cellulase activity is denoted by U. The reducing sugar standard curve needs to be re-plotted each time the DNS solution is changed. The reducing sugar standard curve used in this case is as follows: Figure 10 As shown.

[0058] Determination of crude protein content in culture medium: Take 50 mL of cultured bacterial solution, centrifuge at 10000 r / min for 10 min, discard the supernatant, freeze-dry the bacterial sludge in a freeze dryer, weigh and determine the cell mass; determine the protein content in the freeze-dried bacterial sludge using the Kjeldahl method, calculate the protein yield from the protein content and cell mass, the calculation formula is: protein yield (g / L) = cell mass (g) × protein content (%) / 0.05 × 100 (mL).

[0059] 2. Cultivating lactic acid bacteria in the effluent from citrus processing waste. The citrus processing waste effluent used in this case was prepared and collected in our laboratory (see process details). Figure 11 Store at 4℃ for 1-3 days, or at -20℃ for long-term storage (within 3 months).

[0060] 2.1 Test Methods The lactic acid bacteria culture medium for the citrus processing waste consisted of 500 mL / L citrus processing waste extract, 10 g / L peptone, 2.6 g / L ammonium sulfate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 L of distilled water, adjusted to pH 5.74. The commercial lactic acid bacteria culture medium (MRS broth) consisted of 10 g / L peptone, 8 g / L beef meal, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dimethyl hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, 1 L of distilled water, and a pH of 5.5–5.9. 50 mL of both the citrus processing waste extract lactic acid bacteria culture medium and 50 mL of MRS broth were accurately measured into 150 mL Erlenmeyer flasks and sterilized at 121°C for 15 min. Single colonies of *Lactobacillus plantarum* 28-7 and *Lactobacillus plantarum* 29-3 were picked and cultured in MRS broth overnight at 37°C to prepare primary seed cultures for strains 28-7 and 29-3. The primary seed cultures were then transferred to MRS broth at a ratio of 2% and cultured at 37°C for 18 hours to obtain secondary seed cultures. The secondary seed cultures of 28-7 and 29-3 were then inoculated at a ratio of 2% into lactic acid bacteria culture medium and cultured at 37°C for 5 days. The viable cell count, pH, and lactic acid content in the lactic acid bacteria culture medium were then measured.

[0061] 2.2 Test Results The results of cultivating *Lactobacillus plantarum* 28-7 and *Lactobacillus plantarum* 29-3 in the lactic acid bacteria culture medium prepared from citrus processing waste effluent are shown in Table 4. As shown in Table 4, after 5 days of cultivation using the lactic acid bacteria culture medium prepared from citrus processing waste effluent, the pH of strains 28-7 and 29-3 both decreased to below 4.0, and their lactic acid yields were 19.84 mg / mL and 20.95 mg / mL, respectively. This indicates that citrus processing waste effluent can be used to prepare lactic acid bacteria culture medium, providing energy and carbon for the reproduction of lactic acid bacteria and the synthesis and secretion of lactic acid.

[0062]

[0063] Note: 1) Lactobacillus plantarum 28-7 was screened, preserved and identified by our laboratory. Its Latin name is Lactobacillus plantarum 28-7. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 25, 2019, with the accession number CGMCC17234 and the related patent number ZL202210913907.1; 2) Lactobacillus plantarum 29-3 was screened and identified by our laboratory. Its Latin name is Lactobacillus plantarum 29-3.

[0064] 3. Cultivation of Bacillus in effluent from citrus processing waste 3.1 Test Methods Bacillus has a rich enzyme production system, and the degradation of anti-nutritional factors such as pectin and fiber is a current research hotspot in the field of feed resource development in the livestock industry. Therefore, this experiment aims to investigate the effects of citrus processing waste effluent on the growth and enzyme activity of commonly used Bacillus species.

[0065] The Bacillus spore culture medium for the citrus processing waste consisted of 500 mL / L citrus processing waste extract, 10 g / L peptone, 2.0 g / L ammonium sulfate, 5 g / L sodium chloride, and 1 L of distilled water, adjusted to pH 7.03. The commercial Bacillus spore culture medium (nutrient broth) consisted of 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 1 L of distilled water, and a pH of 7.0–7.4. Accurately measure 70 mL of the Bacillus spore culture medium and 70 mL of the nutrient broth into a 250 mL Erlenmeyer flask, sterilize at 121°C for 15 min, and set aside. Single colonies of *Bacillus belyssioides* DP-2, *Bacillus subtilis* B92, and *Bacillus pumilus* 2Y6 were picked and placed in 250 mL Erlenmeyer flasks containing 70 mL of nutrient broth. The flasks were incubated overnight at 37°C and 220 rpm / min to prepare primary seed culture. These secondary seed cultures were then transferred to the nutrient broth at a 2% concentration and incubated at 37°C and 220 rpm / min for 16 h to obtain secondary seed culture. The secondary seed cultures of strains DP-2, B92, and 2Y6 were inoculated at a 2% concentration into the exfoliated *Bacillus* culture medium and incubated at 37°C and 220 rpm / min for 5 days. The viable cell count, pectinase activity, and cellulase activity were then measured.

[0066] 3.2 Test Results The results of culturing *Bacillus belyssioides* DP-2, *Bacillus subtilis* B92, and *Bacillus pumilus* 2Y6 in the exfoliated *Bacillus* culture medium are shown in Table 5. Table 5 shows that, in terms of viable cell count, compared to nutrient broth, the viable cell counts of strains DP-2 and B92 increased by 45.25 times and 6.10 times, respectively. Regarding pectinase, the pectinase activities in the exfoliated culture medium of strains DP-2, B92, and 2Y6 were 1075.80 U / mL, 676.32 U / mL, and 1932.58 U / mL, respectively, which were 76.39, 9.01, and 63.27 times higher than those in nutrient broth. In terms of cellulase production, no cellulase activity was detected in the nutrient broth medium of strains DP-2, B92, and 2Y6, while the cellulase activities in the exfoliated culture medium were 8.74 U / mL, 14.29 U / mL, and 19.42 U / mL, respectively. This indicates that the sugars in the citrus processing waste effluent can provide energy for the reproduction of Bacillus subtilis, which is beneficial to increasing its viable bacterial count. The citrus residue and pectin in the effluent can act as inducers to stimulate the synthesis and secretion of biological enzymes such as pectinase and cellulase, which is beneficial to improving the activity and yield of biological enzymes. Furthermore, the cost of the Bacillus subtilis culture medium in the effluent is reduced by about 28.21% compared with nutrient broth, indicating that the citrus processing waste effluent has the potential to reduce the cost of industrial fermentation production of enzyme preparations.

[0067]

[0068] Note: 1) Bacillus velezensis DP-2 was screened, preserved and identified by our laboratory. Its Latin name is Bacillus velezensis DP-2. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 25, 2019, with the accession number CGMCC17235 and the related patent number ZL201910302119.7; 2) Bacillus subtilis B92 was screened and identified by our laboratory and is preserved in our laboratory's cryo-store; 3) Bacillus pumilus 2Y6 was screened and identified by our laboratory and is preserved in our laboratory's cryo-store.

[0069] 4. Cultivating yeast in the effluent from citrus processing waste 4.1 Test Methods The yeast extract culture medium consisted of 500 mL / L extract, 10 g / L peptone, 10.5 g / L ammonium sulfate, and 1 L of distilled water, adjusted to pH 6.53. The commercial yeast culture medium (YPD) consisted of 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 1 L of distilled water, and a pH of 6.3–6.7. Accurately measure 80 mL of both the yeast extract culture medium and 80 mL of YPD medium into 250 mL Erlenmeyer flasks, sterilize at 121°C for 15 min, and set aside. Single colonies of *Candida tropicalis* GB3, *Saccharomyces cerevisiae* RA2, and *Callibocae Mayerii* 2120 were picked and cultured overnight at 30°C and 220 rpm to prepare primary seed culture. These were then transferred at a rate of 2% to YPD medium and cultured at 37°C and 220 rpm for 16 hours to obtain secondary seed culture. The secondary seed culture of strains GB3, RA2, and 2120 was inoculated at a rate of 2% into yeast exfoliation medium and cultured at 30°C and 220 rpm for 5 days. The viable cell count, cell mass, and protein content were then measured, and protein yield was calculated.

[0070] 4.2 Test Results As shown in Table 6, in terms of viable cell count, the viable cell counts of *Candida tropicalis* GB3 and *Saccharomyces cerevisiae* RA2 in the yeast extract culture medium were 6.0 × 10⁷ CFU / mL and 3.0 × 10⁷ CFU / mL, respectively, both significantly higher than those in the YPD medium. In terms of cell mass and protein yield, the cell mass and protein yield of *Saccharomyces cerevisiae* RA2 in the yeast extract culture medium were both higher than those in the YPD medium. Furthermore, the cell mass of *Candida tropicalis* GB3 and *Callibik Mayer's yeast* 2120 in the yeast extract culture medium was higher than that in the YPD medium, while the protein yield was lower, indicating that the protein content of *Candida tropicalis* GB3 and *Callibik Mayer's yeast* 2120 grown in the yeast extract culture medium was lower than that in the YPD medium. In addition, compared to the YPD medium, the cost of the yeast extract culture medium was reduced by approximately 28.21%. In summary, this demonstrates that the effluent from citrus processing waste can be used to increase the number of viable yeast cells, increase the protein content of the cells, and produce biological proteins, thereby reducing the production cost of biological proteins and realizing the transformation of low-value waste into high-value products.

[0071]

[0072] Note: 1) Candida tropicalis GB3 was screened and identified by our laboratory. Its Latin name is Candida tropicalis CQGB3. It was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 15, 2024, with the accession number GDMCC64869; 2) Saccharomyces cerevisiae RA2 was screened and identified by our laboratory and is stored in our laboratory's cold storage; 3) Calibick Mayer's yeast 2120 was screened and identified by our laboratory and is stored in our laboratory's cold storage.

Claims

1. A dehydrating agent comprising a main component and minor components, wherein the main component is citric acid, sodium bicarbonate, sodium carbonate, and quicklime, and the minor component is one or more selected from pectinase, cellulase, and xylanase; wherein, The composition ratio of the main component is 1:1~5, the composition ratio of the minor component is 1:1~5, and the ratio of the main component to the minor component is 1:0.02~0.

5.

2. The application of the dehydrating agent as described in claim 1 in the treatment of citrus waste.

3. The method of using the dehydrating agent as described in claim 1, comprising: When citrus waste contains a large amount of citrus peel and the tubers are ≥3cm, crush them to ≤1cm. Mix 0.60~0.86% (W / W) of dehydrating agent with citrus processing waste evenly, let stand for 18~24 hours at below 40℃, and then centrifuge at 2000r / min for 5~10 minutes to separate the solid and liquid. The liquid is the dehydrated liquid and the solid is the dehydrated citrus processing waste.

4. A citrus waste extract, prepared by the method described in claim 3.

5. The application of the extract as described in claim 4 in microbial culture.

6. The application of the extract as described in claim 4 in microbial culture, wherein the microorganisms include, but are not limited to, lactic acid bacteria, Bacillus, and yeast.

7. A microbial culture medium comprising 500 ml / L of the extract as described in claim 4, 10 g / L of peptone, 0-10.5 g / L of ammonium sulfate, water added to a total volume of 1 L, and sodium hydroxide to adjust the pH to 5.5-7.

4.

8. The culture medium of claim 7, comprising: The solution comprises: 500 mL of desalination fluid, 10 g / L peptone, 1.0-3.0 g / L ammonium sulfate, 0.2 g / L magnesium sulfate, and 0.04 g / L manganese sulfate, adjusted to a total volume of 1 L with water, and the pH adjusted to 5.5-5.

9. The microorganism is lactic acid bacteria. Alternatively, the solution comprises: 500 mL / L of desalination fluid, 10 g / L peptone, 0-2.0 g / L ammonium sulfate, and 5.0 g / L sodium chloride, adjusted to a total volume of 1 L with water, and the pH adjusted to 7.0-7.

4. The microorganism is Bacillus. Alternatively, the solution comprises: 500 mL / L of desalination fluid, 10 g / L peptone, 8.5-10.5 g ammonium sulfate, adjusted to a total volume of 1 L with water, and the pH adjusted to 6.3-6.

7. The microorganism is yeast.

9. The culture medium as described in claim 7 or 8, after preparation, is sterilized at 121°C for 15 min, cooled to room temperature, and then inoculated with 2% of the secondary seed culture of the strain according to the volume of the culture medium, wherein the lactic acid bacteria are cultured statically at 37°C, the Bacillus is cultured at 37°C and 220 r / min, and the yeast is cultured at 30°C and 220 r / min.

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