Strain for anaerobic fermentation of cellulosic substrate and use thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a strain for anaerobic fermentation of a cellulose substrate and application thereof.The strain is Lacticaseibacillus chiayiensis SG.X-2 strain, which has been preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO.62423, and the preservation date is April 26, 2022.The application also discloses application of the strain in livestock breeding, and the strain can improve the nutritional value of fiber unconventional feed, improve the growth performance of livestock, and enhance the intestinal barrier function.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain for anaerobic fermentation of cellulose substrate and application thereof. BACKGROUND

[0002] China is a large country in livestock breeding and meat consumption, but the high cost of feed, the abuse of growth-promoting antibiotics and the problem of ecological environmental protection are urgent problems that need to be solved in the livestock breeding industry. In recent years, microbial fermented feed has been more and more widely used in the livestock breeding industry. Microbial fermented feed is a kind of bioactive feed containing probiotics and their metabolites. Microbial fermentation can change the physical and chemical properties of feed substrate and improve the digestibility of feed. At the same time, during the fermentation process, probiotics can be cultured and intermediate metabolites can be provided, thereby maintaining intestinal microbial homeostasis, improving intestinal function and immune function, and ultimately achieving the purpose of feed resource development, animal health improvement and high-quality and safe livestock production. Therefore, the use of microbial fermented feed is an important measure to broaden the channel of feed resources and realize antibiotic-free breeding and healthy and green ecological breeding.

[0003] The energy source of pig and chicken feed is mainly starch carbohydrate such as corn and barley. Although cellulose and starch both belong to carbohydrates, their chemical structures are different. Cellulose is a glucose monomer polymer linked by β-1, 4 glucoside bond. Due to the existence of a large number of hydrogen bonds in or between the molecular chains of natural cellulose, its structure is tight and difficult to be decomposed and utilized. Soybean straw, bran and bran all contain rich cellulose. For a long time, these raw materials have not been reasonably disposed and effectively utilized, most of which are burned or left idle in the field, causing environmental pollution and resource waste. For example, the cellulose in agricultural waste straw is mostly treated by burning, which causes environmental pollution and resource waste. At present, cellulose feed is mainly used for ruminants, and a small amount of cellulose can be added to the diet of breeding pigs according to its fermentation characteristics to improve intestinal health and relieve constipation. In the industrial field, cellulose utilization in China has initially taken shape, and most of them are pretreated by physical or chemical means to change their structure and performance, such as steam explosion, acid or alkali soaking, etc. These processing methods have large energy consumption and serious pollution. In the field of feed, some cellulase products have been used as fermented feed additives at home and abroad. Although the addition of cellulase for degradation is environmentally friendly and efficient, the current cellulase additive is high in dosage and short in time of action in vivo, resulting in unsatisfactory cost and effect.

[0004] Most of the strains currently used for the production of cellulase are wood enzymes, aspergillus and other aerobic bacteria, and some strains are engineering strains, which cannot be directly used for livestock production. During the fermentation process of these aerobic bacteria, cellulose and other carbohydrate substrates are completely degraded to produce carbon dioxide, resulting in a large amount of energy waste. Therefore, the strains currently used for the production of cellulase still have the problems of low functional efficiency and high environmental requirements. SUMMARY

[0005] The present application aims to avoid the shortcomings in the prior art and provides an anaerobic fermentation cellulose substrate strain, which can efficiently ferment and decompose cellulose in an anaerobic environment and produce a variety of functional organic acids to promote animal growth.

[0006] To achieve the above-mentioned application purposes, the following technical solutions are provided:

[0007] The present application provides an anaerobic fermentation cellulose substrate strain, which is Lacticaseibacillus chiayiensis SG.X-2 strain, which has been preserved in Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO.62423 and the preservation date April 26, 2022.

[0008] The source of the above-mentioned anaerobic fermentation cellulose substrate strain is isolated from a high-yield cellulase probiotic strain in pig intestines. The strain can produce cellulase, ferment cellulose and produce a variety of functional organic acids. On the one hand, the strain can ferment cellulose in vitro, and on the other hand, the strain can ferment cellulose substrate to produce functional organic acids, promote the growth and development of livestock and poultry, and promote intestinal health.

[0009] The physiological and biochemical characteristics of the strain are as follows: after culture in a general medium, the single colony is milky white, opaque, rough and wrinkled, and is a gram-positive short rod bacterium.

[0010] The culture medium is: casein enzymatic digest, beef extract powder, yeast extract powder, triammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, glucose, Tween-80, agar, and the volume is made to 1000mL; the pH of the culture medium is 5-6.5.

[0011] The content of the culture medium is as follows: casein enzymatic digest 10.0g, beef extract powder 10.0g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, potassium dihydrogen phosphate 2.0g, glucose 20.0g, Tween-80 1.0g, agar 15g, and the volume is made to 1000mL; the pH of the culture medium is 5.7.

[0012] In the cellulose as the feed raw material substrate, the pH is reduced to about 4.1 after 48h fermentation, the lactic acid production is as high as 33.55mg / mL, the cellulase is 36.25U / g, and the cellulose degradation rate is as high as 82%.

[0013] The application of the strain for anaerobic fermentation of cellulose substrate in the preparation of animal feed is provided.

[0014] The strain can ferment cellulose substrate to produce cellulase and then functional organic acid, and the organic acid can promote the growth and development of livestock and poultry and intestinal health, so that the strain added to the animal feed can effectively improve the growth of animals.

[0015] The application of the strain for anaerobic fermentation of cellulose substrate in the preparation of a reagent for promoting the level of intestinal junction tight protein of animals is provided.

[0016] The application of the strain for anaerobic fermentation of cellulose substrate in the preparation of a reagent for promoting the expression of inflammatory factors is provided.

[0017] Compared with the prior art, the beneficial effects of the present application are:

[0018] The Lacticaseibacillus chiayiensis SG.X-2 strain with high-efficiency fermentation cellulose function is isolated and cultured from the pig intestinal tract, and is obtained by the conventional dilution plate method, the strain has the functions of improving the production performance of animals, enhancing the level of intestinal junction tight protein of animals, and promoting the expression of inflammatory factors, and can be used in animal bodies. BRIEF DESCRIPTION OF DRAWINGS

[0019] The application is further described by using the drawings, but the embodiments in the drawings do not constitute any limitation on the present application, and other drawings can be obtained by the following drawings without creative labor for ordinary skilled in the art.

[0020] Figure 1 is the bacterial sample diagram observed after the strain for anaerobic fermentation of cellulose substrate of the present application is cultured on Congo red medium for 2 days;

[0021] Figure 2 is the colony morphology diagram of the monoclonal strain of the strain for anaerobic fermentation of cellulose substrate of the present application. DETAILED DESCRIPTION

[0022] The application is further described by combining the following examples.

[0023] The source and physiological and biochemical characteristics of the strain for anaerobic fermentation of cellulose substrate of the present application are as follows:

[0024] The strain is Lacticaseibacillus chiayiensis SG.X-2 strain, which has been preserved in Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO.62423, the preservation date April 26, 2022, and the preservation address: No. 59 Building, 5th Floor, Guangzhou, Guangdong.

[0025] The Lacticaseibacillus chiayiensis SG.X-2 strain is obtained by isolating and culturing from pig intestinal tract in September 2020. After enrichment culture of pig intestinal tract microorganisms, 10-fold dilution is performed, and the dilution liquid is diluted to 10 -8 The above dilution liquid (10 -5 ~ 10 -8 ) is coated on Congo red cellulose agar medium plate, and 37℃ culture is performed for 2d, and whether transparent circle is generated is observed. The colony with transparent circle is picked and cultured in ordinary medium at 37℃ anaerobically for 1d.

[0026] The Lacticaseibacillus chiayiensis SG.X-2 strain is observed after being cultured on ordinary medium for 2 days, and the single colony is milk white, opaque, and smooth, as shown in the accompanying Figure 1 gram stain is gram-positive short bacillus, as shown in the accompanying Figure 2 The formula of the ordinary medium is as follows: casein enzymatic digest 10.0g, beef extract powder 10.0g, yeast extract powder 4.0g, ammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, potassium dihydrogen phosphate 2.0g, glucose 20.0g, Tween-80 1.0g, agar 15g, constant volume to 1000mL, and pH is adjusted to 5.7.

[0027] The Lacticaseibacillus chiayiensis SG.X-2 strain is anaerobically cultured in MRS fermentation medium with pH 5.7 at 37℃ for 1 day, and the fermentation liquid is turbid and uniform. The measured 16S rRNA sequence has 700 effective bases, and compared with the 16S rRNA sequence of prokaryotes in GenBank gene library, the 16S rRNA sequence with high sequence homology is called. The strains with similarity of more than 99% are Lacticaseibacillus chiayiensis, and the strain is identified as Lacticaseibacillus chiayiensis combined with morphological and molecular biological characteristics.

[0028] Example 1 Fermentation characteristics of Lacticaseibacillus chiayiensis SG.X-2

[0029] Lacticaseibacillus chiayiensis SG.X-2 was inoculated into the cellulose medium at 10 7 CFU / mL, and the pH was detected at 4h, 12h, 24h and 48h of fermentation, respectively. The cellulase activity, lactic acid content and residual cellulose were analyzed by sampling, and the cellulose degradation rate was calculated. The results are shown in Table 1.

[0030] Table 1 Fermentation characteristics of Lacticaseibacillus chiayiensis SG.X-2

[0031]

[0032] It was found that the pH decreased to about 4.1 after 48h of fermentation, the lactic acid yield was as high as 33.55mg / mL, the cellulase reached 36.25U / g, and the cellulose degradation rate was as high as 82%. It can be seen that the fermentation characteristics of Lacticaseibacillus chiayiensis SG.X-2 are good, the lactic acid (organic acid) yield is high, and the cellulase yield is high.

[0033] Example 2 Effect of Lacticaseibacillus chiayiensis SG.X-2 fermented feed on the growth performance of white-feathered broilers

[0034] 120 one-day-old white-feathered broilers were selected and randomly divided into 3 groups according to body weight, with 4 replicates in each group and 10 broilers in each replicate. The control group (CON) was fed with the basic diet, the fermented fiber feed 0.2kg / T group (0.2kg / T) was fed with the basic diet + 0.2kg / T fermented fiber feed, and the fermented fiber feed 5kg / T group (5kg / T) was fed with the basic diet + 5kg / T fermented fiber feed. After 3 weeks, the growth performance indicators of white-feathered broilers were measured, and the results are shown in Table 1.

[0035] Table 2 Effect of Lacticaseibacillus chiayiensis SG.X-2 fermented fiber feed on the growth performance of broilers

[0036]

[0037] Note: Different lowercase letters indicate significant differences (P < 0.05), and no annotation or the same lowercase letter indicates no significant difference (P > 0.05); the same below. Control, control group; 0.2 kg / t, 200 ppm group; 5 kg / t, 5000 ppm group; the same below. BW, body weight gain; ADFI, average daily feed intake.

[0038] The results showed that the daily weight gain of the fermented feed group was significantly higher than that of the control group, and the feed utilization efficiency was significantly improved, indicating that the addition of fermented feed containing Lacticaseibacillus chiayiensis SG.X-2 in the basal diet can improve the growth performance of broilers, and Lacticaseibacillus chiayiensis SG.X-2 can promote the level of intestinal tight junction protein in animals.

[0039] Example 3 Effect of Lacticaseibacillus chiayiensis SG.X-2 in Drinking Water on the Intestinal Health of White-feathered Broilers

[0040] Based on the production performance results, the intestinal health indicators of broilers were further detected. It was found that the addition of fermented fiber feed could significantly reduce the content of intestinal MDA. In addition, the results of fluorescent quantitative PCR showed that the mRNA expression of IFN-α in the intestinal mucosa of the ileum of broilers in the 5 kg / T group was significantly lower than that of the control group, and the expression of IL-4 was significantly higher than that of the control group; in terms of intestinal barrier, the mRNA expression of ZO-1 and ZO-2 in the 5 kg / T group was significantly higher than that of the control group, while the expression of Claudin-2 was significantly lower, and the results are shown in Table 3.

[0041] Table 3 Effect of Lacticaseibacillus chiayiensis SG.X-2 Fermented Fiber Feed on the Intestinal Health of Broilers

[0042]

[0043] Note: IFN-α, Interferon-α; MDA, serum malondialdehyde; IL-4, interleukin-4; ZO-1, tight junction protein-1; ZO-2, tight junction protein-2; Claudin-2, occludin-2.

[0044] This indicates that the addition of fermented feed in the basal diet can alleviate intestinal oxidative damage and enhance intestinal barrier function, indicating that Lacticaseibacillus chiayiensis SG.X-2 can promote the expression of inflammatory factors.

[0045] Example 4 Effect of fermented fiber feed with Lacticaseibacillus chiayiensis SG.X-2 on growth performance of piglets

[0046] 120 three-way cross weaned piglets with similar body weight were selected and randomly divided into 3 groups, 4 replicates per group, 10 pigs per replicate. Three days of pre-feeding were conducted before the start of the experiment, and the experiment lasted for 28 days. The control group (CON) was fed with the basal diet, the fermented feed 0.2 kg / T group (0.2 kg / T) was fed with the basal diet + 0.2 kg / T fermented feed, and the fermented feed 5 kg / T group (5 kg / T) was fed with the basal diet + 5 kg / T fermented feed. The results are shown in Table 4.

[0047] Table 4 Effect of fermented fiber feed with Lacticaseibacillus chiayiensis SG.X-2 on growth performance of piglets

[0048]

[0049] Note: ADG, average daily gain; ADFI, average daily feed intake; F:G, feed conversion ratio.

[0050] The above results show that the growth performance indicators and diarrhea rate of piglets were measured after 4 weeks. It was found that the fermented feed in the 5 kg / T group can significantly improve the daily gain of piglets, reduce the feed conversion ratio and the diarrhea rate. This indicates that the addition of fermented feed in the basal diet can improve the growth performance of piglets.

[0051] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A strain for anaerobic fermentation of cellulose substrates, characterized in that: The strain is *Lactobacillus chiayiense* ( Lacticaseibacillus chiayiensis The strain SG.X-2 has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.62423 and deposit date of April 26, 2022.

2. The strain for anaerobic fermentation of cellulose substrate according to claim 1, characterized in that, The physiological and biochemical characteristics of the strain are as follows: After being cultured on a normal culture medium, its single colonies are milky white, opaque, with a rough and wrinkled surface, and are Gram-positive short bacilli.

3. The strain for anaerobic fermentation of cellulose substrate according to claim 2, characterized in that, The culture medium consists of: casein digest, beef extract powder, yeast extract powder, triammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, potassium dihydrogen phosphate, glucose, Tween-80, and agar, diluted to 1000 mL; the pH of the culture medium is 5-6.

5.

4. The strain for anaerobic fermentation of cellulose substrate according to claim 3, characterized in that, The culture medium contains the following components: 10.0g casein digest, 10.0g beef extract, 4.0g yeast extract, 2.0g triammonium citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 2.0g potassium dihydrogen phosphate, 20.0g glucose, 1.0g Tween-80, and 15g agar, diluted to 1000mL; the pH of the culture medium is 5.

7.

5. The strain for anaerobic fermentation of cellulose substrate according to claim 1, characterized in that, Using cellulose as the feed substrate, after fermentation for 48 hours, the pH decreased to about 4.1, the lactic acid production reached 33.55 mg / mL, the cellulase content reached 36.25 U / g, and the cellulose degradation rate reached 82%.

6. The use of a strain of anaerobic fermentation cellulose substrate as described in any one of claims 1 to 5 in the preparation of animal feed.

7. The use of a strain of anaerobic fermentation cellulose substrate according to any one of claims 1 to 5 in the preparation of a reagent that promotes the level of intestinal tightness protein in animals.

8. The use of a strain of anaerobic fermentation cellulose substrate according to any one of claims 1 to 5 in the preparation of a reagent that promotes the expression of inflammatory factors.

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