A solid-state fermentation feed additive and its preparation method and application

Abstract

The present invention discloses a solid-state fermentation feed additive and a preparation method and application thereof, and relates to the field of biotechnology. The solid-state fermentation feed additive includes a glutamine-producing methylbacterium radiation-resistant, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 8, 2024, with a preservation address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No: 64329. The methylbacterium radiation-resistant has the ability to produce high glutamine, and can use wheat bran and tea residues as the main raw materials to produce glutamine by solid-state fermentation, and the glutamine yield can reach 70g / kg. The fermentation culture prepared by solid-state fermentation of the methylbacterium radiation-resistant with wheat bran and tea residues as the main raw materials can be used as a feed additive. It can be applied to fish farming, which can significantly improve the intestinal digestive enzyme activity and antioxidant capacity of fish.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a solid-state fermentation feed additive and a preparation method and application thereof. Background Art

[0002] Glutamine is a conditionally essential amino acid. When the synthesis in the animal body is insufficient, it needs to be taken from the outside to maintain the stability of the glutamine content in the body. As a conditionally essential amino acid, glutamine has the functions of improving immune function, enhancing resistance, promoting protein synthesis, and protecting intestinal function. Studies have shown that glutamine can significantly increase the absorption of leucine and proline in the intestine of grass carp, and can significantly increase the anabolism of intestinal protein. In addition, adding glutamine to the feed can significantly improve the phagocytic ability of leukocytes, respiratory burst activity and nonspecific immunity of grouper, loach and young carp.

[0003] At present, glutamine is mostly produced by liquid deep fermentation of Corynebacterium glutamicum followed by separation and purification. Fermentation production has high equipment requirements and complex production processes, resulting in high prices for glutamine. At present, glutamine in feed is mainly obtained by external addition, and the premixing process is complex, resulting in high cost of use in feed.

[0004] The present invention intends to produce glutamine by microbial fermentation of commonly used feed raw materials, thereby reducing the cost of glutamine in feed, significantly improving the intestinal digestive enzyme activity and antioxidant capacity of grass carp, and improving the intestinal tissue morphology and structure, thereby providing new ideas for the development of safe and efficient feed or additives. Summary of the invention

[0005] The purpose of the present invention is to provide a solid-state fermentation feed additive and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The solid-state fermentation feed additive comprises a glutamine-producing Methylobacterium radiotolerans, which can produce glutamine by solid-state fermentation with wheat bran and tea residue as main raw materials, and the glutamine yield can reach 70g / kg.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a glutamine-producing radiation-resistant methylbacterium, which was deposited in Guangdong Provincial Microbial Culture Collection Center on May 8, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No: 64329.

[0008] The present invention also provides the use of the radioresistant Methylobacterium in the fermentation production of glutamine.

[0009] Furthermore, the fermentation is solid-state fermentation.

[0010] The present invention also provides the use of the radioresistant methylbacterium in the preparation of a solid-state fermentation feed additive.

[0011] The present invention also provides a method for preparing a solid-state fermentation feed additive, comprising the steps of inoculating the radioresistant methylbacterium into a solid-state fermentation medium and preparing the solid-state fermentation feed additive through solid-state fermentation.

[0012] Furthermore, the solid-state fermentation medium comprises the following components: 50-200 g / kg wheat bran, 200-500 g / kg tea residue, 0.5-3 g / kg dipotassium hydrogen phosphate, 0.3-0.8 g / kg magnesium sulfate and 0.0001-0.01 g / kg ferrous sulfate.

[0013] The present invention also provides a solid-state fermentation feed additive prepared according to the above preparation method.

[0014] The present invention also provides application of the solid-state fermentation feed additive in preparing fish breeding feed.

[0015] The present invention also provides a fish breeding feed, comprising the solid-state fermentation feed additive.

[0016] The present invention also provides application of the solid-state fermentation feed additive or fish farming feed in fish farming.

[0017] The present invention discloses the following technical effects:

[0018] The radiotolerant Methylobacterium (named HZ-01) provided by the present invention has the ability to produce high glutamine. The strain can use wheat bran and tea residue as main raw materials to produce glutamine by solid-state fermentation, and the glutamine yield can reach 70g / kg.

[0019] The fermentation culture of Methylobacterium radiotolerant HZ-01, which is prepared by solid-state fermentation with wheat bran and tea residue as main raw materials, can be used as a feed additive. When applied to fish farming, it can significantly improve the intestinal digestive enzyme activity and antioxidant capacity of fish and improve the intestinal tissue morphology and structure, thus providing a new idea for the development of safe and efficient feed or additives. The present invention also lays an important foundation for the in-depth study and utilization of glutamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 these drawings without paying creative work.

[0021] Figure 1 This is a graph showing changes in glutamine production during the fermentation process of strain HZ-01;

[0022] Figure 2 This is a microscope observation picture of strain HZ-01;

[0023] Figure 3 This is the colony morphology of strain HZ-01;

[0024] Figure 4 Observation pictures of the intestinal tissue morphology and structure of grass carp in different treatment groups; ae are sections of the intestinal tissue morphology and structure of grass carp in groups J0, J1, J2, J3 and J4, respectively; the boxes represent the rupture and damage of intestinal villi. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The culture medium formula used in the following examples is as follows:

[0031] GAXA-1 solid culture medium: 20 g / L high-gluten flour, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L ferrous sulfate and 20 g / L agar powder, adjust the pH to 7.3.

[0032] GAXA-1 liquid culture medium: 20g / L high-gluten flour, 0.5g / L dipotassium hydrogen phosphate, 0.5g / L magnesium sulfate and 0.01g / L ferrous sulfate, adjust the pH to 7.3.

[0033] GAXA fermentation medium (1kg): 100g wheat bran, 450g tea residue (Iron Guanyin), 0.5g potassium dihydrogen phosphate (0.5-3g can achieve the same effect), 0.5g magnesium sulfate (0.3-0.8g can achieve the same effect) and 0.001g ferrous sulfate (0.0001-0.01g can achieve the same effect), and water is added to 1kg.

[0034] Example 1

[0035] 1. Isolation of strains

[0036] The bottom mud collected from small shed shrimp breeding ponds in Taishan City, Guangdong Province in May 2023 was screened for microorganisms on GAXA-1 solid culture medium using a 10-fold serial gradient dilution separation method.

[0037] The strain screened on GAXA-1 solid medium was transferred to GAXA-1 liquid medium and cultured at 30°C and 150 r / min for 48 hours to obtain seed liquid. After that, the seed liquid was inoculated into GAXA fermentation medium at a 5% inoculation amount and fermented at 30°C and 150 r / min for 72 hours.

[0038] After 72 hours of fermentation, the number of live bacteria and the content of glutamine in the fermentation medium (the detection method is "GB5009.124-2016") were detected, and finally the strain HZ-01 with high live bacteria count and strong glutamine production ability was screened out for solid-state fermentation using wheat bran and tea residue (Iron Guanyin) as substrates. Figure 1 The graph shows the change of glutamine production of strain HZ-01 during the fermentation process. The results show that the strain can accumulate the glutamine content to 7wt% within 72h, proving that the strain has excellent ability to produce glutamine.

[0039] 2. Strain Identification

[0040] 2.1 Morphological identification and detection of physiological and biochemical characteristics

[0041] The screened strain HZ-01 was inoculated onto a GAXA-1 solid culture medium plate and cultured at 30°C for 72 h. The colony growth was observed, and the bacteria were picked to prepare smears for Gram staining. The bacterial morphology and spore formation were observed under a microscope for morphological identification.

[0042] Figure 2 This is a microscope observation picture of strain HZ-01. Figure 3 The colony morphology of strain HZ-01 is shown in Figure 1. The results show that the colony of strain HZ-01 is round, with irregular edges and opaque; the size of a single cell is (0.3-0.5) microns × (2.0-2.2) microns; it is Gram-negative; and there are no spores.

[0043] 2.2 Molecular Biological Identification

[0044] The strain HZ-01 was inoculated in GAXA-1 liquid medium with an inoculation volume of 5% (V / V), a temperature of 33°C, a shaking speed of 150 r / min, and cultured for 24 hours. The bacterial genome was extracted using a DNA extraction kit (purchased from Beijing Biotech Biotechnology Co., Ltd.). The 16S rRNA gene was amplified by PCR using the bacterial genome as a template, and the amplified product was sent to the Guangdong Provincial Microbiological Culture Collection Center for sequencing analysis.

[0045] The nucleotide sequence of 16S rRNA of strain HZ-01 obtained is shown in SEQ ID NO.1.

[0046] SEQ ID NO.1:

[0047]

[0048] The 16S rRNA sequence was compared on NCBI (https: / / blast.ncbi.nlm.nih.gov) and found that the homology between strain HZ-01 and Methylobacterium radiotolerans reached 99.92%.

[0049] Comparison on EZBioCloud (https: / / www.ezbiocloud.net) showed that strain HZ-01 had a homology of 99.92% with Methylobacterium radiotolerans, 99.61% with Methylobacterium tardum, and 99.38% with Methylobacterium oryzae.

[0050] The strain HZ-01 was identified as Methylobacterium radiotolerans.

[0051] 3. Strain preservation

[0052] The screened radiation-resistant Methylobacterium HZ-01 was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 8, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No: 64329.

[0053] Example 2

[0054] Preparation method of solid-state fermentation feed additive:

[0055] The HZ-01 strain was inoculated into the GAXA-1 liquid medium, and cultured at 30°C and 150 r / min for 48 hours to obtain a seed solution. After that, the seed solution was inoculated into the GAXA fermentation medium at an inoculation amount of 5%, and fermented and cultured at 30°C and 150 r / min for 72 hours to obtain a solid fermentation product, which was then subjected to low-temperature drying to obtain a solid fermentation feed additive.

[0056] Comparative Example 1

[0057] According to the same method as in Example 1, commercially available radioresistant Methylobacterium (Beina Biotechnology Co., Ltd.) was inoculated into GAXA-1 liquid culture medium and cultured at 30°C and 150 r / min for 48 hours to obtain seed liquid. After that, the seed liquid was inoculated into GAXA fermentation medium at an inoculum of 5%, and fermented at 30°C and 150 r / min for 72 hours. After 72 hours of fermentation, the glutamine content in the fermentation medium was detected.

[0058] The results showed that after 72 hours, the glutamine content in the fermentation medium of this comparative example was 0.6 wt %.

[0059] Example 3

[0060] In 2022, product verification tests will be conducted in the Guangdong Nutriera Group's recirculating aquaculture system, as follows:

[0061] 1 Materials and methods

[0062] 1.1 Experimental feed

[0063] The experiment set up 5 treatment groups, with J0 group as the control group and J1-D4 as the experimental groups. The control group was fed with a basic feed, and the experimental group was fed with an alternative feed, which was prepared by replacing the soybean meal in the basic feed with the solid fermentation feed additive prepared in Example 2, and the replacement mass ratios of J1-D4 were 3%, 6%, 9% and 12% respectively.

[0064] The basic feed formula used in each treatment group is shown in Table 1. The feed raw materials were crushed by Lida superfine grinder and passed through a 40-mesh sieve. Various raw materials were accurately weighed according to the formula requirements. After mixing, a double-screw plodder was used to extrude granular feed with a particle size of 2.0 mm. After air-drying in a cool place, it was stored at -20°C for future use.

[0065] Table 1 Basic feed formula 1

[0066]

[0067] Note: 1. The crude protein content of the basic feed is 28.09% and the crude fat content is 4.50%.

[0068] 2. Each kg of herbivorous premix contains: Vitamin A 350000IU, Vitamin D 380000IU, Vitamin E 2500mg, Vitamin K 3320mg, Vitamin B1 400mg, Vitamin B2 430mg, Vitamin B6 420mg, Vitamin B 123.0mg, vitamin C 3500mg, D-calcium pantothenate 1250mg, niacinamide 2500mg, folic acid 250mg, D-biotin 6.0mg, inositol 3000mg; magnesium sulfate 600mg, zinc methionine 1800mg, manganese sulfate 1100mg, copper sulfate 1050mg, glycinate iron 11000mg, cobalt sulfate 120mg, calcium iodate 90mg and sodium selenite 25mg.

[0069] 1.2 Experimental animals and feeding management

[0070] Grass carp fry were purchased from a seed farm in Zhongshan City, and the breeding experiment was carried out in the Guangdong Nutriera recirculating breeding system (the volume of the circulating filtration aquarium was about 300L). The grass carp fry were temporarily raised for 2 weeks before the start of the experiment, and basic feed was fed during this period. At the beginning of the formal experiment, 450 healthy, lively and scratch-free grass carps were selected and randomly divided into 5 treatment groups, with 3 replicates in each group and 30 fish in each replicate. The initial weight of the grass carp fry was 5.52±0.09g. The experiment lasted for 8 weeks. During the experiment, the basic feed or alternative feed was fed twice a day (8:00 and 16:00) according to the group, and the daily feeding amount was 4% to 6% of the fish body weight. The feeding amount was adjusted according to the actual feeding situation, and the water was changed once every 2 days. During the experiment, the ammonia nitrogen concentration in the water was ≤0.20mg / L, and the water temperature was (18.5±2.6)℃.

[0071] 1.3 Sample collection and determination

[0072] 1.3.1 Growth index determination

[0073] At the end of the breeding experiment, the animals were fasted for 24 hours, counted, weighed, and their growth indicators were measured, including weight gain rate, feed coefficient, feeding rate, survival rate, organ-to-body ratio, liver-to-body ratio, and fatness. The calculation formulas for each indicator are as follows:

[0074] Weight gain rate (WGR, %) = (Wt-W0) / W0×100;

[0075] Feed coefficient ratio (FCR) = F / (Wt total + W1 - W0 total);

[0076] Daily feed intake (DFI, % / d) = F / [t×(Wt total+W1+W0 total) / 2]×100;

[0077] Survival rate (SR, %) = final number / initial number × 100;

[0078] Viscerosomatic index (VSI, %) = Wi / Wh × 100;

[0079] Hepatosomatic index (HSI, %) = Wy / Wh × 100;

[0080] Condition factor (CF, %) = Wh / L 3 ×100;

[0081] In the above calculation formula, W0-average weight at the beginning of the experiment (g); W1-weight of death during the experiment (g); Wt-average weight at the end of the experiment (g); d-breeding days; F-feeding amount (g); t-breeding time (d); W0total-total weight at the beginning of the experiment (g); Wttotal-total weight at the end of the experiment (g); Wi-visceral mass (g); Wyliver mass (g); Wh-grass carp weight (g); L-grass carp body length (cm).

[0082] 1.3.2 Determination of intestinal digestive enzyme activity and antioxidant capacity

[0083] After the breeding experiment, stop feeding for 24 hours, randomly select 5 fish from each barrel, use a 1.0mL sterile syringe to collect blood from the tail vein, place it in a 1.5mL centrifuge tube, let it stand overnight at 4℃, centrifuge it at 3000r / min for 10min, take the supernatant and mix it in a 0.5mL sterile centrifuge tube, and store it at -80℃. Randomly select 3 grass carps from each tank, quickly separate the intestinal tissue, wash it with physiological saline, put it in a 1.5mL enzyme-free centrifuge tube, and store it at -80℃ for later use.

[0084] The contents of trypsin, lipase, amylase, creatine kinase (CK), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and reduced glutathione (GSH) in the intestine of grass carp were determined using kits from Nanjing Jiancheng Bioengineering Institute.

[0085] 1.3.3 Preparation and observation of intestinal tissue sections

[0086] After the breeding experiment, the fish were stopped from feeding for 24 hours. One fish was randomly selected from each barrel and its intestine was taken out. After removing the mesentery and fat attachments around the intestinal tissue, the hind intestine of each fish was quickly cut out and placed in a pre-prepared 4% paraformaldehyde fixative. The fish were fixed for 24 hours, dehydrated with conventional alcohol, embedded in paraffin sections, and then prepared for histological observation and observed under a microscope.

[0087] 1.4 Data Statistical Analysis

[0088] SPSS 20.0 software was used for one-way analysis of variance, and Duncan's multiple comparisons were performed. P < 0.05 was considered to be significantly different; P > 0.05 was considered to be insignificant. All data are expressed as mean ± standard error.

[0089] 2 Experimental results and analysis

[0090] 2.1 Effects of solid-state fermentation feed additives on growth performance of grass carp

[0091] The growth index test results of each treatment group are shown in Table 2. As shown in Table 2, compared with the J0 group, the weight gain rate of grass carp in the J1 group was significantly reduced (P < 0.05), but the survival rate of grass carp in each treatment group was improved, and the survival rate of the J3 and J4 groups was significantly improved (P < 0.05), and there was no significant difference in the indicators of grass carp in other groups (P > 0.05).

[0092] Table 2 Growth index test results

[0093]

[0094]

[0095] 2.2 Effects of solid-state fermentation feed additives on intestinal enzyme activities of grass carp

[0096] The results of intestinal digestive enzyme activity detection in each treatment group are shown in Table 3. As shown in Table 3, compared with the J0 group, the intestinal trypsin and lipase activities of grass carp in each treatment group were increased, among which the trypsin activity was significantly increased (P < 0.05), and the intestinal creatine kinase activity of grass carp in the J3 group was significantly increased (P < 0.05).

[0097] Table 3 Results of intestinal digestive enzyme activity test

[0098]

[0099] The results of intestinal antioxidant capacity test of each treatment group are shown in Table 4. As shown in Table 4, compared with group J0, the superoxide dismutase activity and glutathione peroxidase activity of grass carp in each treatment group showed an upward trend, and the activity of group J4 was the highest (P>0.05), and the reduced glutathione content of grass carp in the intestine of each treatment group was significantly reduced (P<0.05); there was no significant difference in the intestinal malondialdehyde content among the groups (P>0.05).

[0100] Table 4 Antioxidant capacity test results

[0101]

[0102] 2.3 Effects of solid-state fermentation feed additives on the morphological structure of grass carp intestinal tissue

[0103] The results of observation on the intestinal tissue morphology of grass carp in each treatment group are as follows: Figure 4 As shown. Figure 4 It can be seen that compared with the J0 group, the intestinal villi of the grass carp in the J1 group were sparse and the villi were broken and damaged. Using the solid-state fermentation feed additive of the present invention, the intestinal villi damage phenomenon of the J2 and J3 groups was improved, but the arrangement was not neat; the arrangement and density of the J4 intestinal villi were improved, and the villi outline was clear. In general, the intestinal muscle layer thickness of the J1 group, J2 group, J3 group and J4 group was better than that of the J0 group.

[0104] In summary, adding the solid-state fermentation feed additive prepared by the present invention to grass carp feed can improve the growth performance, intestinal digestive enzyme activity and antioxidant capacity of grass carp, and improve the intestinal tissue morphology and structure, wherein the effect is best at a 9% addition level.

[0105] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A glutamine-producing strain of Methylobacterium radiotolerans, characterized in that: The radiation-resistant Methylobacterium was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 8, 2024, with the storage address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No: 64329.

2. Use of the Methylobacterium radiotolerant strain according to claim 1 in fermentation production of glutamine.

3. The use according to claim 2, characterized in that: The fermentation is solid state fermentation.

4. Use of the Methylobacterium radiotolerant strain according to claim 1 in preparing a solid-state fermentation feed additive.

5. A method for preparing a solid-state fermentation feed additive, characterized in that: The method comprises the steps of inoculating the radioresistant methylbacterium according to claim 1 into a solid-state fermentation medium, and preparing the solid-state fermentation feed additive through solid-state fermentation.

6. The preparation method according to claim 5, characterized in that: The solid-state fermentation medium comprises the following components: 50-200 g / kg of wheat bran, 200-500 g / kg of tea dregs, 0.5-3 g / kg of dipotassium hydrogen phosphate, 0.3-0.8 g / kg of magnesium sulfate and 0.0001-0.01 g / kg of ferrous sulfate.

7. A solid-state fermentation feed additive prepared according to the preparation method according to claim 5 or 6.

8. Use of the solid-state fermentation feed additive according to claim 7 in preparing fish farming feed.

9. A fish farming feed, characterized in that: Including the solid-state fermentation feed additive according to claim 7.

10. Use of the solid-state fermentation feed additive according to claim 7 or the fish farming feed according to claim 9 in fish farming.

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