Application of fructo-oligosaccharide in rumen fluid cryopreservation and fruit residue anaerobic fermentation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of fructo-oligosaccharide in rumen fluid cryopreservation and fruit residue anaerobic fermentation. The fructo-oligosaccharide is used as a cryoprotectant for long-term cryopreservation of rumen fluid. According to the application method of the fructo-oligosaccharide, the microbial activity of rumen fluid can be effectively maintained, the cellulose degradation efficiency can be improved in the fruit residue fermentation process, and a new technical means is provided for efficient utilization of biomass resources. When the collected rumen fluid is subjected to cryopreservation, toxicity to microorganisms is avoided, and the situation that the rumen fluid cannot be stored for a long time is avoided. According to the invention, the problem that the rumen fluid cannot be preserved for a long time due to toxicity of 2-methyl sulfoxide to microorganisms in a method for cryopreserving the collected rumen fluid in the prior art is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to the application of oligofructose in the cryopreservation of rumen fluid and the anaerobic fermentation of fruit residues. Background Art

[0002] As the global demand for renewable energy and environmental protection continues to increase, research on the use of biomass resources to produce clean energy and high value-added products has received widespread attention. Fruit pomace is a large amount of by-products produced during the processing of fruits such as juice and jam. It is rich in polysaccharides such as cellulose, hemicellulose and pectin, and is a biomass resource with potential utilization value. Converting fruit pomace into volatile fatty acids, methane and other products through fermentation can not only reduce the environmental pressure of waste treatment, but also achieve efficient utilization of resources. Among them, the English name of volatile fatty acids is Volatile Fatty Acid, abbreviated as VFA in English. The chemical name of methane is CH4.

[0003] Rumen fluid is a complex microbial system derived from the rumen contents of ruminants. It is rich in a variety of cellulose-degrading enzymes and other beneficial microorganisms and plays an important role in biomass fermentation, especially in the degradation of refractory cellulose. However, the microorganisms in rumen fluid are extremely sensitive to environmental conditions, and their activity and stability will be significantly affected under low-temperature storage conditions. Therefore, how to effectively protect the activity of microorganisms in rumen fluid during long-term storage and transportation has become an urgent problem to be solved.

[0004] Fructo-oligosaccharides are naturally occurring functional oligosaccharides with good water solubility and biocompatibility, and are widely used in food and medicine. Fructo-oligosaccharides are also known as FOS.

[0005] At present, the most common method for collecting rumen fluid for anaerobic fermentation of fruit residues is to collect fresh rumen fluid from ruminants directly from pastures or slaughterhouses. However, if it is far away from pastures or slaughterhouses, it will be inconvenient to collect. Therefore, current research tends to collect rumen fluid and freeze it. Commonly used rumen fluid preservation methods include: refrigerating at 4°C or freezing at -20°C without adding any protective agent, and adding cryoprotectants such as 2-methyl sulfoxide or glycerol to preserve rumen fluid. However, rumen fluid can only be stored for a short period of time without adding cryoprotectants, not more than 7 days, otherwise the microbial activity and anaerobic fermentation efficiency will be significantly reduced. The cryoprotective effect of adding glycerol is poor, not as good as 2-methyl sulfoxide. Therefore, the 2-methyl sulfoxide currently used for cryopreservation of collected rumen fluid is toxic to microorganisms, which will cause the rumen fluid to be unable to be stored for a long time. Summary of the invention

[0006] In order to solve the problem that 2-methyl sulfoxide used in the prior art for cryopreservation of collected rumen fluid is toxic to microorganisms and causes the rumen fluid to be unable to be preserved for a long time, the present invention aims to develop a method for long-term preservation of rumen fluid using oligofructose as a cryoprotectant and for fermenting fruit pomace. The present invention provides the use of oligofructose in cryopreservation of rumen fluid and anaerobic fermentation of fruit pomace.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] The invention provides an application of oligofructose in cryopreservation of rumen fluid. The oligofructose is used as a cryoprotectant for long-term cryopreservation of rumen fluid.

[0009] The molecular weight of the oligofructose is 180.15588, and its molecular formula is C6H 12 O6, the structural formula is as follows:

[0010]

[0011] The present invention provides the use of oligofructose as a cryoprotectant for long-term storage of rumen fluid. The present invention does not cause toxicity to microorganisms during cryopreservation of the collected rumen fluid, thereby preventing the rumen fluid from being unable to be stored for a long time. The present invention solves the problem that 2-methyl sulfoxide used in the prior art method for cryopreservation of collected rumen fluid is toxic to microorganisms, causing the rumen fluid to be unable to be stored for a long time.

[0012] The oligofructose provided by the present invention is used as a cryoprotectant, and the storage time of rumen fluid is 3 months when used for long-term storage. The existing methods for freezing and storing rumen fluid are mostly stored at -80°C, which has a high storage cost and is for short-term storage, such as within 1 week to 1 month. Compared with the existing methods for freezing and storing rumen fluid, the storage time of rumen fluid is extended and can be used for long-term storage of rumen fluid.

[0013] Preferably, after the oligofructose is added to the rumen fluid, rumen fluid added with oligofructose is prepared; and after the oligofructose-added rumen fluid is cryopreserved, rumen fluid cryopreserved with oligofructose is obtained.

[0014] Preferably, the temperature of the frozen storage is -18° C. to -20° C. At this temperature, the rumen fluid can be stored for a long time and the microbial activity of the rumen fluid can be effectively maintained.

[0015] Preferably, the frozen storage period is 80 to 100 days.

[0016] Preferably, the mass ratio of the fructo-oligosaccharide to the rumen fluid added with fructo-oligosaccharide is 4% to 6%.

[0017] Preferably, the mass ratio of fructo-oligosaccharide to rumen fluid in the rumen fluid added with fructo-oligosaccharide is 3.5% to 6.5%.

[0018] Preferably, the rumen fluid is rumen fluid collected from the rumen of cattle.

[0019] Preferably, the rumen fluid is rumen fluid collected from the rumen of healthy cows equipped with permanent rumen fistula.

[0020] The present invention also provides the use of the rumen fluid frozen and preserved by oligofructose in anaerobic fermentation of fruit residues. The rumen fluid added with oligofructose is prepared by using the oligofructose as a cryoprotectant, and then the rumen fluid frozen and preserved by oligofructose is obtained after freezing. The rumen fluid frozen and preserved by oligofructose is added to the fruit residue sample, and the rumen fluid is fermented and cultured at a constant temperature of 37°C to 0°C for 72h to 96h in an anaerobic environment.

[0021] After fermentation, the fermentation liquid was filtered and the residue was collected to determine the degradation rate of nutrients; at the same time, the total volatile fatty acid concentration and methane production produced during the fermentation process were evaluated.

[0022] The mass ratio of the fruit pomace sample to the volume of the rumen fluid cryopreserved with oligofructose is 1kg:70L-80L.

[0023] Preferably, the mass ratio of the fruit pomace sample to the volume of the rumen fluid cryopreserved with oligofructose is 1 kg:73 L to 77 L.

[0024] The present invention uses oligofructose as a cryoprotectant to preserve rumen fluid for a long time. The rumen fluid can be preserved for 3 months at -20°C. The frozen rumen fluid is used in fruit pomace fermentation. The application method provided by the present invention can not only effectively maintain the microbial activity of rumen fluid, but also improve the cellulose degradation efficiency during the fruit pomace fermentation process, providing a new technical means for the efficient utilization of biomass resources.

[0025] Preferably, the rumen fluid frozen and stored with oligofructose is thawed before being added to the fruit residue sample for fermentation, that is, the rumen fluid frozen and stored with oligofructose is thawed before fermentation.

[0026] Preferably, the thawing method is as follows:

[0027] First equilibrate at 3℃~5℃ for 25 minutes to 35 minutes, then thaw at 38℃~40℃.

[0028] Preferably, the fruit pomace sample is a powder obtained by first drying the fruit pomace, then crushing it, and passing it through a 1 mm to 2 mm sieve.

[0029] Preferably, the fruit pomace is any one of citrus pomace, apple pomace and grape pomace.

[0030] Preferably, the drying temperature is 60°C-65°C.

[0031] Preferably, a 200 mesh nylon bag is used for filtration.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention provides the use of oligofructose in the cryopreservation of rumen fluid and the anaerobic fermentation of fruit residues. The present invention provides the use of oligofructose as a cryoprotectant for the long-term preservation of rumen fluid. The present invention will not be toxic to microorganisms when the collected rumen fluid is cryopreserved, and will not cause the rumen fluid to be unable to be preserved for a long time. The present invention solves the problem that the method of cryopreserving the collected rumen fluid in the prior art uses 2-methyl sulfoxide, which is toxic to microorganisms and causes the rumen fluid to be unable to be preserved for a long time.

[0034] 2. The present invention uses oligofructose as a cryoprotectant to preserve rumen fluid for a long time. The rumen fluid can be preserved for 3 months at -20°C. The frozen rumen fluid is used in fruit pomace fermentation. The application method provided by the present invention can not only effectively maintain the microbial activity of rumen fluid, but also improve the cellulose degradation efficiency during the fermentation of fruit pomace, providing a new technical means for the efficient utilization of biomass resources.

[0035] 3. The results of the present invention show that compared with fresh rumen fluid, the results of volatile fatty acids and methane produced by rumen fluid frozen with 5%wt oligofructose added and stored at -20°C for 3 months are less different. The use of frozen rumen fluid significantly reduced the production of volatile fatty acids and methane, and the difference was obvious compared with fresh inoculum. From the perspective of the microbiome, the rumen microbial composition and functional characteristics related to carbohydrate metabolism verified the results of volatile fatty acid and methane production. Therefore, the use of rumen fluid frozen with oligofructose for up to 3 months at -20°C is a reliable and cost-effective method for preserving fruit residue anaerobic digestion inoculum, especially when it is difficult to obtain fresh rumen fluid, which is beneficial to reduce the need for laboratory maintenance of donor animals: fistula ruminants, or reduce the frequency of collecting rumen fluid from slaughterhouses. In future studies, this technology needs to be verified by longer fermentations in anaerobic digestion using various substrates and anaerobic digestion systems.

[0036] Among them, the English abbreviation of fresh rumen fluid is FRE; the English abbreviation of rumen fluid protected by oligofructose is FOS; the English abbreviation of volatile fatty acids is VFA; and the chemical structural formula of methane is CH4. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 For the alpha diversity analysis of rumen microorganisms in the present invention; wherein, Figure 1 Figure a in the figure is the ACE index; Figure 1 Figure B is the Shannon index; FRE is fresh rumen fluid; FOS is rumen fluid protected by fructooligosaccharide cryoprotection; FRO is frozen rumen fluid.

[0038] Figure 2 This is a clustering tree at the genus level for rumen microbial samples in the present invention; wherein, FRE is fresh rumen fluid; FOS is rumen fluid protected by cryo-fructose oligosaccharide; and FRO is frozen rumen fluid.

[0039] Figure 3 is the effect of the rumen fluid preservation method of the present invention on the relative abundance (%) of microbial groups related to the methanogenic process; wherein, Figure 3 The a figure in the figure is the phylum: Ciliophora; Figure 3 The genus in the figure b is Entodinium; Figure 3 The c figure in the figure is the genus: Isotricha; Figure 3 The genus in the d figure is Methanobrevibacter; ***P<0.001; FRE is fresh rumen fluid; FOS is fructooligosaccharide-free rumen fluid; FRO is frozen rumen fluid.

[0040] Figure 4 For the cluster analysis of carbohydrate enzymes in the present invention; wherein, Figure 4 Figure a in the figure is a hierarchical clustering tree of carbohydrate-active enzymes at the family level; Figure 4 Figure b is a hierarchical clustering tree of carbohydrate-active enzymes in the glycoside hydrolase class; among them, FRE is fresh rumen fluid; FOS is rumen fluid protected by fructooligosaccharide cryoprotection; FRO is frozen rumen fluid; GH is glycoside hydrolase. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1

[0043] 1. Experimental Methods

[0044] 1. Citrus residue treatment

[0045] 1 g of citrus pulp was dried in an oven at 60 °C for 48 hours, and then ground with a Chinese herbal medicine grinder until it passed a 2 mm sieve to obtain a citrus pulp sample for chemical composition determination. The specific chemical components determined included dry matter, ash, crude protein, ether extract, starch, neutral detergent fiber, acid detergent fiber and acid detergent lignin, crude fat and organic matter.

[0046] Among them, citrus residue was provided by Baoji Liupanyun Biotechnology Co., Ltd.

[0047] The method for determining the dry matter in the citrus residue sample is as follows: drying the citrus residue sample prepared above at 105° C. to constant weight, weighing its mass, and determining the dry matter content of the citrus residue sample.

[0048] The method for determining the ash content in citrus pulp samples refers to GB / T 6438-2007 Determination of crude ash in feed.

[0049] The difference between the dry matter content in the citrus pulp sample and the ash content in the citrus pulp sample is the organic matter content in the citrus pulp sample.

[0050] The method for determining crude protein in citrus pulp samples refers to the national standard "GB / T 6432-2018 Determination of crude protein in feed - Kjeldahl method".

[0051] The method for determining crude fat in citrus pulp samples refers to the national standard “GB / T 6433-2006 Determination of crude fat in feed”.

[0052] The determination method of neutral detergent fiber in citrus pulp samples refers to the national standard GB / T20806-2022 Determination of neutral detergent fiber (NDF) in feed. The determination method of acidic detergent fiber in citrus pulp samples refers to the reference standard NY / T 1459-2022 Determination of acidic detergent fiber in feed. The determination method of acidic detergent lignin in citrus pulp samples refers to the national standard GB / T 20805-2006 Determination of acidic detergent lignin (ADL) in feed.

[0053] The dry matter content of the citrus pulp sample was determined to be 95.41%, organic matter was 89.43%, crude protein was 9.59%, crude fat was 3.64%, neutral detergent fiber was 26.77%, acid detergent fiber was 12.09%, and acid detergent lignin was 2.88%.

[0054] Among them, the English abbreviation of neutral detergent fiber is NDF; the English abbreviation of acid detergent fiber is ADF.

[0055] 2. Collection and pretreatment of rumen fluid

[0056] First, rumen fluid was collected from the rumen of three healthy donor cows equipped with permanent rumen fistulas. The donor cows were milked three times a day. Each donor cow was fed 25 kg of total mixed diet per day. Feeding was carried out at 7:00 and 15:00 every day, and water and food were available freely. Rumen fluid was collected 1 hour before morning feeding, filtered through 4 layers of gauze, and the rumen fluid of the three donor cows was mixed evenly in equal volumes, placed in a thermos bottle, and quickly transported back to the Beijing Key Laboratory of Dairy Cow Nutrition, and placed in a constant temperature water bath at 39°C for insulation, and CO2 was continuously introduced for standby use.

[0057] Among them, the donor dairy cows came from the Yanqing Breeding Farm of Beijing Shounong Animal Husbandry Development Co., Ltd. The breed was Chinese Holstein cow, with an average weight of 682±17.2kg and an average milk production of 34.5±1.7kg / d.

[0058] The formula of the total mixed ration for the donor dairy cows is as follows: 34.59% whole corn silage, 11.85% alfalfa hay, 4.33% oat hay, 17.73% crushed corn kernels, 3.98% flaked corn, 10.46% soybean meal, 2.90% rapeseed meal, 0.56% beet pellets, 7.12% whole cottonseed, 1.33% fat powder, 1.28% molasses, and 3.87% premix. All the above values ​​are mass ratios based on dry matter.

[0059] 3. Experimental design

[0060] Three samples were used for three treatments: fresh rumen fluid, frozen rumen fluid, and frozen rumen fluid with 5%wt oligofructose added. Among them, the English name of fresh rumen fluid is FRE; the English name of frozen rumen fluid is FRO; the English name of frozen rumen fluid with 5%wt oligofructose added is FOS.

[0061] Fresh rumen fluid was used immediately after collection, whereas FRO and FOS samples were equilibrated at 4°C for 30 min before being stored at -20°C for 90 days. Prior to fermentation, samples from each frozen treatment were taken and thawed in a 39°C water bath.

[0062] It should be noted that the frozen rumen fluid added with 5%wt oligofructose, i.e., the rumen fluid frozen and stored with oligofructose, is prepared by adding 5%wt oligofructose to the rumen fluid, and then freezing and storing it.

[0063] Among them, oligofructose was purchased from Beijing Solebow Biotechnology Co., Ltd.

[0064] Artificial saliva, also known as buffer solution, was prepared by weighing 0.013 g CaCl2·2H2O, 0.001 g CoCl2·6H2O, 0.010 g MnCl2·4H2O, 0.008 g FeCl3·6H2O, 0.832 g NH4HCO3, 7.280 g NaHCO3, 1.966 g Na2HPO4·12H2O, 0.125 g MgSO4·7H2O, 1.290 g KH2PO4, 0.390 g L-cysteine ​​hydrochloride, 0.390 g Na2S·9H2O, and 0.1 mg resazurin on an analytical balance, dissolving in 1 L of distilled water and mixing evenly. The above reagents were purchased from Sinopharm Group and were analytically pure.

[0065] Weigh 2g of citrus pulp sample into a 125mL anaerobic fermentation bottle equipped with a chitin stopper and a Heinz screw cap. Add 50mL of the above buffer and 25mL of rumen fluid, and pass N2 into the bottle to remove air. Plug in the stopper and tighten the bottle cap, connect the fermentation bottles one by one to the AGRS-Ⅲ microbial fermentation micro-gas production automatic recorder in sequence, and automatically record the real-time gas production. Set up 6 replicates for each treatment. All fermentation bottles were cultured continuously in a 39℃ constant temperature biochemical incubator for 96h. During the fermentation process, shake manually every 2 hours to ensure that the fermentation substrate and inoculation solution are evenly mixed. Repeat the culture 3 times.

[0066] After 96 hours of fermentation, turn off the gas production recording system. Pour all the fermentation liquid in the bottle into a dried and weighed 200-mesh nylon bag for filtration. Use a pH meter to measure the pH of the filtrate. Collect the filtrate and freeze it at -20°C for subsequent analysis and testing. Rinse the nylon bag containing the residue with tap water until it is clear. Place the collected residue together with the nylon bag in a 65°C oven and dry it continuously for 48 hours until constant weight. After drying, transfer the nylon bag to a dryer, cool it, and weigh it. Determine the content of dry matter, neutral detergent fiber, and acid detergent fiber in the residue, and calculate the degradation rate of citrus residue dry matter, neutral detergent fiber, and acid detergent fiber.

[0067] Among them, pH meter: Leichi PHS-3E, Shenzhen, China.

[0068] The English abbreviation for dry matter degradation rate is DMD; the English abbreviation for neutral detergent fiber degradation rate is NDFD; the English abbreviation for acid detergent fiber degradation rate is ADFD.

[0069] 4. Indicator analysis

[0070] 4.1 Analysis of gas production indicators

[0071] The gas production data recorded by the AGRS-Ⅲ microbial fermentation micro-gas production automatic recorder were fitted with the gas production curve by the NLIN program of SAS 9.4:

[0072] V t =V max ×{1-e [-Kg×(T-L)]};

[0073] Where, T is time (h), V t is the real-time in vitro fermentation gas production (mL), V max is the theoretical maximum gas production (mL), Kg is the gas production rate (mL / h), and L is the delay time when gas production starts (h).

[0074] 4.2 Calculation of dry matter, neutral detergent fiber and acid detergent fiber degradation rates

[0075] DMD(%)=100×(W0–W t ) / (W0–W k );

[0076] Where DMD is the dry matter degradation rate; W0 is the fermentation substrate: the initial dry matter of citrus residue, W t is the dry matter of the residue after fermentation, W k It is the dry matter of fermentation residue in blank group.

[0077] NDFD(%)=100×(W0×NDF%–W t ×NDF%) / W0×NDF%;

[0078] Wherein, NDFD is the degradation rate of neutral detergent fiber; W0 is the fermentation substrate: the initial neutral detergent fiber of citrus pulp, W t It is the neutral detergent fiber after fermentation residue, W k It is the neutral detergent fiber of the fermentation residue of the blank group.

[0079] ADFD(%)=100×(W0×ADF%–W t ×ADF%) / W0×ADF%;

[0080] Wherein, ADFD is the acid detergent fiber degradation rate. W0 is the fermentation substrate: the initial acid detergent fiber of citrus pulp, W t It is the acidic detergent fiber after fermentation residue, W k It is the acidic detergent fiber of the fermentation residue of the blank group.

[0081] 4.3 Sample index analysis

[0082] The volatile fatty acid content in the samples was measured using a gas chromatograph. A flame ionization detector and a capillary column were used during the analysis, with an injector temperature of 200°C, a detector temperature of 220°C, and a column temperature of 110°C.

[0083] The total volatile fatty acid concentration was calculated as acetate + propionate + butyrate + isobutyrate + valerate + isovalerate + 2-methylbutyrate, where 2-methylbutyrate co-eluted with isovalerate under this analytical condition.

[0084] Among them, gas chromatograph: 7820AGC, Agilent Technologies.

[0085] Capillary column: DB-FFAP; 30 m × 0.25 mm × 0.25 μm; Agilent Technologies.

[0086] The English abbreviation for total volatile fatty acid concentration is TVFA.

[0087] Meanwhile, the concentrations of CH4 and CO2 in the 96-h gas samples were determined by gas chromatograph using a capillary column and a thermal conductivity detector; the temperatures of the injector, detector and column box were 45°C, 110°C and 80°C, respectively.

[0088] Among them, gas chromatograph: 7820A GC, Agilent Technologies.

[0089] Capillary column: GS-CarbonPLOT, 30 m × 0.32 mm × 3.00 μm; Agilent Technologies.

[0090] 4.4 Rumen microbial analysis

[0091] DNA was extracted from rumen fluid using a DNA extraction kit. TM 2000c spectrophotometer and agarose gel electrophoresis. Among them, DNA extraction kit: Stool DNAKit, Omega Bio-Tek.

[0092] To prepare the sequencing library, the extracted DNA was fragmented into 400 bp and sequenced using NEXTFLEX TM The library was constructed using Rapid DNA-Seq Kit.

[0093] Sequencing was performed on the Illumina Hiseq X Ten platform. For the preprocessed read sequences, MEGAHIT version 1.2.9 was used to assemble contigs (>200 bp), and MetaGene was used to predict protein-coding genes in contigs. A non-redundant gene catalog was established by CD-HIT based on 95% sequence similarity and 90% coverage. The gene catalog was aligned with the non-redundant protein sequence database and the carbohydrate-active enzyme database using Diamond version 0.8.35 to obtain species annotations and carbohydrate-active enzyme information, with an E value cutoff of E<1×10-5 To control the false-positive rate, the P values ​​were corrected using the Benjamini-Hochberg method.

[0094] Among them, Illumina Hiseq X Ten platform: 2×150bp; Illumina.

[0095] The English abbreviation of non-redundant protein sequence database is NR.

[0096] The English abbreviation of the Carbohydrate Active Enzyme Database is CAZy.

[0097] The English abbreviation for carbohydrate active enzymes is CAZymes.

[0098] 2. Results

[0099] 1. Nutrient degradation rate and volatile fatty acid yield after 96h of fermentation

[0100] Results As shown in Table 1, there were no significant differences in the degradation rates of dry matter, neutral detergent fiber, and acid detergent fiber of citrus pulp between the FOS group and the FRE group. However, the degradation rates of dry matter and neutral detergent fiber of citrus pulp in the FRO group were reduced compared with those in the FRE group (P<0.05). In addition, the concentrations of total volatile fatty acids, acetic acid, and propionic acid in the FRO group were lower than those in the FRE and FOS groups (P<0.05). Compared with the FRE group, the FOS group did not affect the production of isobutyric acid, butyric acid, isovaleric acid, 2-methylbutyric acid, or valeric acid.

[0101] The above results indicated that the microorganisms in the rumen fluid of the FOS group were still better than those in the rumen fluid of the FRE group in decomposing the cellulose in citrus pulp, thereby producing more VFA.

[0102] Table 1. Effects of rumen fluid storage methods on nutrient degradation rate and volatile fatty acid production

[0103]

[0104] Note: DMD is the dry matter degradation rate; NDFD is the neutral detergent fiber degradation rate; ADFD is the acid detergent fiber degradation rate; VFA is the volatile fatty acid. 2 FRE is fresh rumen fluid; FOS is rumen fluid protected by oligofructose cryoprotection; FRO is frozen rumen fluid. Different letters in the same row in a and b indicate significant differences, P < 0.05.

[0105] 2. Gas production and gas production fitting parameters during fermentation

[0106] Comparison of gas production and fitting parameters is shown in Table 2. The total gas production and maximum gas production (Vmax) were the highest in fresh rumen fluid within 96 h. Mechanical damage to the cell structure due to cold shock, which caused changes in microbial concentrations, may be the reason for the reduced gas production when using preserved rumen fluid. However, the gas production at 96 h was not significantly different between the FRE and FOS groups (P>0.05). From 24 h to 96 h, higher cumulative gas production was recorded in the fermented citrus pulp in the FOS group, compared with lower production in the FRE group (P<0.05).

[0107] The above research results showed that the FOS group could be used as an inoculum source for anaerobic fermentation.

[0108] Inoculation with rumen fluid from different preservation methods did not affect the percentage of H2. Rumen fluid from the FOS group had no significant effect on the percentage of CH4 compared to the FRE group (P>0.05). However, the percentage of CO2 was higher and the percentage of CH4 was lower in the FRE group compared to the other two treatments (P<0.05). This may indicate that methanogens are very sensitive to the cryopreservation and thawing process, which may be due to the reduction in the number of ciliates caused by freezing. Because the concentration of ciliates is linearly correlated with the amount of CH4 emission. In this study, the FOS group increased the percentage of CH4, indicating that the use of FOS cryoprotection is an effective method to maintain the population or activity of methanogens in rumen fluid.

[0109] Table 2. Effects of rumen fluid storage methods on gas production and gas production fitting parameters

[0110]

[0111] Note: 1 DM is dry matter; Vmax is the theoretical maximum gas production; Kg is the gas generation rate; 2 FRE is fresh rumen fluid; FOS is rumen fluid protected by cryo-fructose oligosaccharide; FRO is frozen rumen fluid; different letters in the same row in a and b indicate significant differences, P < 0.05.

[0112] 3. Rumen microbial structure after 96 hours of fermentation

[0113] A total of 896,452,915 raw sequences were obtained from 18 samples, with an average of 49,802,939 sequences per sample and a standard deviation of 5,467,403. After quality control, 905,433,767 sequences without host contamination were generated, with an average of 50,301,876 sequences per sample and a standard deviation of 5,434,238. The Alpha diversity parameter was used to determine the richness and diversity of the microbial community. Compared with the FOS group, the ACE and Shannon indices of the FRE group decreased, as shown in Figure 2. Figure 1 Figure a and Figure 1 As shown in Figure b.

[0114] The results showed that long-term cryopreservation would destroy the richness and diversity of microorganisms, while the FOS group could effectively protect rumen microbial cells.

[0115] Among them, Alpha diversity parameters: ACE and Shannon index.

[0116] As shown in Table 3, at the phylum level, the relative abundance of rumen microorganisms in the FRE group and the FOS group was closer. For example, the abundance of Firmicutes, Bacteroidetes, Actinobacteria, and Euryarchaeota in the FRE group and the FOS group was not significantly different, but all of them were significantly different from the FRO group. At the genus level, the dendrogram analysis of the hierarchical clustering analysis was performed using the unweighted paired arithmetic mean method. It was found that the samples in the FRE group and the FOS group were closely clustered at the genus level, such as Figure 2 shown.

[0117] The above results indicate that the FOS group had higher similarity in microbial composition with the FRE group than with the FRO group.

[0118] Among them, the English abbreviation of unweighted paired arithmetic mean method is UPGMA.

[0119] Table 3. Effect of rumen fluid storage method on the abundance of rumen microbial phyla (top 10) after 96h of fermentation

[0120]

[0121] Note: 1 FRE is fresh rumen fluid; FOS is rumen fluid protected by cryo-fructose oligosaccharide; FRO is frozen rumen fluid; different letters in the same row in a and b indicate significant differences, P < 0.05.

[0122] Ciliated protozoa can contribute up to 50% of the microbial biomass in rumen fluid, and Entodinium has been reported to be the dominant genus (Lan and Yang, 2019). Although rumen ciliated protozoa themselves cannot synthesize methane, they are important symbionts associated with methane production because they are H2 producers and carry a large number of associated methanogens (Hook et al., 2010). In the present invention, we observed that the abundance of Ciliophora, Entodinium, and Isotricha in the FRO group was reduced compared with the FRE group, while these protozoan groups did not differ significantly between the FRE and FOS groups, as shown in Table 1. Figure 3 Figure a in - Figure 3 Similar differences were observed in Methanobrevibacter, as shown in Figure c. Figure 3 d in Figure 3. Previous studies have shown that Entomophthora and Isotrichum genera play a role in supporting methanogenesis, which may be related to the higher activity of their hydrogenosomes (Newbold et al., 2015). Therefore, the mutualistic relationship between hydrogen-producing protozoa and methanogens may explain the changes in methane production. From the perspective of the microbiome, our results show that frozen rumen fluid stored at -20°C using FOS as a long-term cryoprotectant has little effect on methanogenesis during citrus pulp fermentation.

[0123] To calculate the rumen microbial carbohydrate enzyme similarity / distance between treatments, we also constructed a dendrogram based on UPGMA analysis. The UPGMA cluster tree also showed that at the family level of carbohydrate-active enzymes, samples from the FRE and FOS groups were close to each other, showing a clustering pattern similar to that of the microbiota composition, such as Figure 4 As shown in Figure a. It is well known that GH enzymes break glycosidic bonds in carbohydrate molecules. Similarly, at the class level of GH, the distance between the FRE group and the FOS group is also very close, as shown in Figure 5. Figure 4 As shown in Figure b.

[0124] Table 4. Carbohydrate-active enzymes with significant differences at the family level (top 30) (%)

[0125]

[0126] Note: 1 FRE is fresh rumen fluid; FOS is rumen fluid protected by cryo-fructose oligosaccharide; FRO is frozen rumen fluid; different letters in the same row in a and b indicate significant differences, P < 0.05.

[0127] Table 4 shows the carbohydrate-active enzymes that changed significantly among the treatments. GH enzymes include cellulases, endohemicellulases, debranching enzymes, and oligosaccharide degrading enzymes, which are diverse catalytic modules involved in the cell wall extension process (Suryawanshi et al., 2019). GH2 and GH3 are abundant oligosaccharide degrading enzymes with broad substrate preferences, such as β-glucosidase, α-L-arabinofuranosidase, and β-galactosidase.

[0128] In the present invention, the abundance of GH2 and GH3 was not significantly different between the FRE group and the FOS group. However, GH2 and GH3 were significantly reduced in the FRO group compared with the FRE group. Similar results were also observed in the relative abundance of GH31, GH51, GH13, GH115, GH78, GH92, GH36 and GH26.

[0129] The above results show that FOS can protect the carbohydrate enzyme activity of microorganisms to a certain extent. In addition, CE (esterase) plays a vital role in the degradation of pectin and hemicellulose, and contributes to the complete hydrolysis of plant polysaccharides (Kabel et al., 2011). Pectin is abundant in citrus peel. CE10 and CE12 are key enzymes for degrading pectin. In the present invention, the abundance of CE10 and CE12 in the FOS group was similar to that in the FRE group, indicating that the addition of FOS can effectively protect pectinase-producing bacteria during the long-term storage of rumen fluid at -20°C.

[0130] The results for volatile fatty acids and methane produced by the FOS group were less different compared to the FRE group. The FRO group showed significantly reduced volatile fatty acids and methane production, while the difference was clear compared to the FRE group. From a microbiome perspective, the rumen microbial composition and functional traits related to carbohydrate metabolism validated the results for volatile fatty acids and methane production. Therefore, the storage of rumen fluid using FOS at -20°C for up to 3 months is a reliable and cost-effective method to preserve inoculum for anaerobic digestion of fruit pomace, especially in cases where access to fresh rumen fluid is difficult, which is beneficial to reduce the need for laboratory maintenance of donor animals: fistulated ruminants, or to reduce the frequency of collecting rumen fluid from slaughterhouses. In future studies, this technology needs to be validated with longer fermentations in anaerobic digestion using various substrates and anaerobic digestion systems.

[0131] From the above results, it can be seen that the present invention provides an application of oligofructose in the cryopreservation of rumen fluid and anaerobic fermentation of fruit residues. The research results of the present invention show that oligofructose has the effect of protecting the activity of rumen microorganisms for a long time. By adding FOS as a cryoprotectant, the present invention effectively protects the activity of microorganisms in rumen fluid, so that the rumen fluid can be stored for up to 3 months at -20°C, maintaining the diversity and abundance of microorganisms, and ensuring the activity of microorganisms during anaerobic digestion.

[0132] The present invention provides the use of oligofructose as a cryoprotectant for long-term storage of rumen fluid. The present invention does not cause toxicity to microorganisms when cryopreserving the collected rumen fluid, thereby preventing the rumen fluid from being unable to be stored for a long time. The present invention solves the problem that in the prior art method of cryopreserving the collected rumen fluid, 2-methyl sulfoxide is toxic to microorganisms, causing the rumen fluid to be unable to be stored for a long time.

[0133] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. The application of oligofructose in cryopreservation of rumen fluid is characterized in that: The oligofructose is used as a cryoprotectant for long-term cryopreservation of rumen fluid.

2. The use according to claim 1, characterized in that: After the oligofructose is added to the rumen fluid, rumen fluid added with oligofructose is prepared; after the oligofructose-added rumen fluid is cryopreserved, rumen fluid cryopreserved with oligofructose is obtained.

3. The use according to claim 2, characterized in that: The freezing storage temperature is -18°C to -20°C.

4. The use according to claim 2, characterized in that: The frozen storage time is 80 to 100 days.

5. The use according to claim 2, characterized in that: The mass ratio of the oligofructose to the rumen fluid added with oligofructose is 4% to 6%.

6. The use according to claim 5, characterized in that: The mass ratio of oligofructose to rumen fluid in the rumen fluid added with oligofructose is 3.5% to 6.5%.

7. The use according to claim 2, characterized in that: The rumen fluid is rumen fluid collected from the rumen of cattle.

8. Use of the rumen fluid frozen and preserved with oligofructose according to claim 2 in anaerobic fermentation of fruit residues.

9. The use according to claim 8, characterized in that: Adding the rumen fluid frozen and preserved with oligofructose to the fruit pomace sample, and fermenting under anaerobic environment; The mass ratio of the fruit pomace sample to the volume of the rumen fluid cryopreserved with oligofructose is 1kg:70L-80L.

10. The use according to claim 9, characterized in that: The rumen fluid frozen and preserved with oligofructose is thawed and then added to the fruit pomace sample; The thawing method is as follows: First equilibrate at 3℃~5℃ for 25 minutes to 35 minutes, then thaw at 38℃~40℃.

Images