Compound lactic acid bacterium agent for producing yak feed by fermenting highland barley vinasse as well as application and application method of compound lactic acid bacterium agent

Abstract

The invention belongs to the technical field of fermented feed, and particularly relates to a compound lactic acid bacterium agent for producing yak feed by fermenting highland barley vinasse, and application and an application method thereof. The invention provides a compound lactic acid bacterium agent for producing yak feed by fermenting highland barley vinasse, which is prepared by compounding bacterium liquid of lactobacillus plantarum, bacterium liquid of lactobacillus buchneri, bacterium liquid of lactobacillus fermentum and bacterium liquid of pediococcus pentosaceus according to the volume ratio of (2-4): (1-3): (1-3): (0.5-2). Cellulose components in the raw materials such as the highland barley vinasse, the alfalfa hay and the rapeseed cake can be fully decomposed, so that the utilization rate of the raw materials is greatly improved, and meanwhile, the content of nutritional ingredients such as crude protein in the fermented feed and the oxidation resistance of the feed are improved; a new way is provided for development of the yak industry and improvement of the additional value of the highland barley vinasse.

Description

Technical Field

[0001] This invention belongs to the field of fermented feed technology, specifically relating to a compound lactic acid bacteria agent for the fermentation of highland barley lees to produce yak feed, its application, and application method. Background Technology

[0002] Barley, an annual herbaceous plant belonging to the Poaceae family, is characterized by its strong cold resistance, short growth cycle, high yield, early maturity, and wide adaptability. As a result, it has become a dominant crop in the Qinghai-Tibet Plateau and surrounding high-altitude and cold regions. At present, its application has expanded from a traditional food crop to a complete industrial chain of staple food, processing, and by-product resource utilization. It is a key carrier for agricultural economy and ecological cycle in high-altitude and cold regions.

[0003] Currently, besides being used as a staple food crop, the largest processing method for highland barley is its use as a raw material for brewing highland barley wine. This process generates a large amount of highland barley lees, which is the core byproduct of the current highland barley industry chain. Due to its high moisture content, short shelf life, and limited sales radius, highland barley lees are highly susceptible to spoilage if not handled properly. Of course, simply composting the lees can also lead to increased microbial populations, toxin accumulation, and alcohol residue, damaging the soil and water environment. Although highland barley lees are high in fiber and protein, they still suffer from an unbalanced amino acid profile, making them difficult to digest and resulting in low utilization rates. Therefore, improving the utilization rate of highland barley lees is of paramount importance.

[0004] Fermented feed, especially lactic acid bacteria fermented feed, has become a core direction for improving the quality of yak feed due to its ability to degrade crude fiber and increase protein content, as disclosed in patents such as CN114601041A, CN117958359A, and CN109793101A. However, the process of preparing fermented feed using lactic acid bacteria still faces the following key technical bottlenecks: Insufficient strain compatibility leads to low nutrient conversion efficiency in feed. Although the technology for preparing fermented feed using lactic acid bacteria is relatively mature at present, if a single lactic acid bacteria is used for fermentation, it cannot fully degrade the crude fiber of raw materials such as highland barley distillers' grains, and can only slightly increase the protein content, resulting in poor fermentation efficiency. If multiple lactic acid bacteria are used for compound fermentation, although the fermentation efficiency can be improved to some extent, it still cannot meet the dual requirements of improving nutritional quality and enhancing immunity.

[0005] Therefore, considering the characteristics of highland barley distillers' grains and combining them with local raw materials such as alfalfa hay, a high-performance microbial agent for fermentation should be developed. By improving the fermentation process, the utilization of highland barley distillers' grains can be realized. At the same time, the problems of high crude fiber content, poor palatability, and high content of anti-nutritional factors such as tannins in local forage should be solved. The goal is to prepare a fermented feed suitable for yaks, which is of great significance for the development of yak breeding industry in high-altitude and cold regions and the reuse of highland barley distillers' grains by-product resources. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a compound lactic acid bacteria agent for the fermentation of highland barley lees to produce yak feed, its application, and its application method.

[0007] The technical solution provided by this invention is as follows: The first aspect of the present invention is to provide a compound lactic acid bacteria agent for the fermentation of highland barley lees to produce yak feed. The compound lactic acid bacteria agent includes Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus fermentum, and Pediococcus pentosaceus. The compound lactic acid bacteria agent is obtained by first culturing each strain into a bacterial solution, and then combining the bacterial solutions of Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus fermentum, and Pediococcus pentosaceus in a volume ratio of 2~4:1~3:1~3:0.5~2. In the compound lactic acid bacteria inoculant, the viable count of each lactic acid bacteria solution is 1.0 × 10⁻⁶. 9 cfu / mL ~1.0×10 11 cfu / mL.

[0008] Preferably, in the compound lactic acid bacteria inoculant, the volume ratio of *Lactobacillus plantarum*: *Lactobacillus brunelli*: *Lactobacillus fermentum*: *Pediococcus pentosus* is 3~4:2:2:1.

[0009] Preferably, the compound lactic acid bacteria agent is prepared by the following method: S1 strain activation: Pick single colonies from the freeze-dried tubes and inoculate each single colony into MRS liquid medium, and incubate at 25~45℃ for 12~24h. S2 expansion culture: Inoculate the activated bacterial solution from S1 into the expansion culture medium at an inoculum rate of 0.5%~2% and expand the culture until the bacterial solution OD reaches the target value. 600 When the pH reaches 1.8–2.0, centrifuge, collect the bacterial sludge, wash 2–3 times with sterile physiological saline, and resuspend until the viable cell count in the bacterial culture reaches 1.0 × 10⁻⁶. 9 cfu / mL ~1.0×10 11 cfu / mL; S3 bacterial solution compounding: Mix the bacterial solutions of Lactobacillus plantarum, Lactobacillus bruneri, Lactobacillus fermentum and Pediococcus pentosaceus in a volume ratio of 3~4:2:2:1 to obtain the compound lactic acid bacteria inoculum.

[0010] A second aspect of the present invention is the application of the aforementioned compound lactic acid bacteria agent in the preparation of yak feed from fermented highland barley lees.

[0011] A third aspect of the present invention is to provide a specific method for applying the compound lactic acid bacteria agent in the preparation of yak feed from fermented highland barley lees, comprising the following steps: (1) Take barley lees, add alfalfa powder, barley bran, rapeseed cake, beet pulp, calcium carbonate and brown sugar to it, stir evenly, add water during stirring to adjust the moisture content of the material to 40%~60%, and pre-wet at room temperature for 12~24 hours; By weight, the ingredients are: 60-80 parts highland barley lees, 10-20 parts alfalfa hay powder, 5-15 parts highland barley bran, 5-10 parts rapeseed cake, 1-5 parts beet pulp, 0.5-1.5 parts calcium carbonate, and 1-2 parts brown sugar. (2) Spray the compound lactic acid bacteria agent evenly into the material prepared in (1) at an inoculation rate of 2%~8% (v / w) and stir thoroughly to mix evenly; (3) After inoculation, the material is put into a sealed fermentation tank, the air is exhausted, and fermentation is carried out at 20~28℃ for 2~3 days. During the fermentation process, the pH value is monitored and dipotassium hydrogen phosphate is added to maintain the pH at 5.5~6.0. (4) After fermentation, the material is transferred to a drying equipment and dried at 40~45℃ until the moisture content of the material is ≤12%. After drying, it is crushed to 4~5mm, packaged and sealed to obtain the yak feed.

[0012] The beneficial effects of this invention are as follows: (1) A compound lactic acid bacteria agent for the fermentation of barley lees to produce yak feed is provided. The compound lactic acid bacteria agent is composed of bacterial solutions of Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus fermentum and Pediococcus pentosus in a volume ratio of 2~4:1~3:1~3:0.5~2. It can fully decompose the cellulose components in raw materials such as barley lees, alfalfa hay, and rapeseed cake, greatly improving the utilization rate of raw materials and increasing the content of nutrients such as crude protein in fermented feed. (2) The fermented feed made by fermenting barley lees with the above-mentioned compound lactic acid bacteria agent contains rich antioxidant components, such as glutathione (GSH) with a maximum content of 19.4 mg / g. In addition, the DPPH free radical scavenging rate of the feed is between 64.2% and 74.3%. The increase in the content of these active ingredients is beneficial to the improvement of yak disease resistance to a certain extent. Furthermore, after feeding the above-mentioned feed to yaks, their daily weight gain is 1.42 to 1.52 kg / d, and their growth performance is significantly improved. Detailed Implementation

[0013] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0014] Example 1 Yak feed was prepared by fermenting highland barley distiller's grains with a compound lactic acid bacteria agent. The specific preparation method is as follows: (1) Take 70 parts of barley lees, add 15 parts of alfalfa powder, 10 parts of barley bran, 8 parts of rapeseed cake, 2 parts of beet pulp, 1 part of calcium carbonate and 1 part of brown sugar, stir evenly, add water during stirring to adjust the moisture content of the material to 50%, and pre-wet at room temperature for 24 hours. (2) Spray the compound lactic acid bacteria agent evenly into the material prepared in (1) at an inoculation rate of 4% (v / w) and stir thoroughly to mix evenly; In this embodiment, the compound lactic acid bacteria inoculant is prepared by the following method: S1 strain activation: Single colonies were picked from the freeze-dried tubes and inoculated into MRS liquid medium and cultured at 35°C for 24 hours. S2 Scale-up Culture: The activated bacterial solution from S1 was inoculated into the scale-up medium at an inoculation rate of 1% (v / v) for scale-up culture. After centrifugation, the bacterial sludge was collected, washed three times with sterile physiological saline, and resuspended until the viable cell count in the bacterial solution was 1.0 × 10⁻⁶. 9 cfu / mL; S3 bacterial solution compounding: The bacterial solutions of Lactobacillus plantarum, Lactobacillus bruneri, Lactobacillus fermentum and Pediococcus pentosaceus are mixed evenly in a volume ratio of 4:2:2:1 to obtain the compound lactic acid bacteria inoculum. (3) After inoculation, the material is put into a sealed fermentation tank, the air is exhausted, and fermentation is carried out at 25°C for 3 days. During the fermentation process, the pH value is monitored and dipotassium hydrogen phosphate is added to maintain the pH at 5.8~6.0. (4) After fermentation, the material is transferred to a drying equipment and dried at 45°C until the moisture content of the material is ≤12%. After drying, it is crushed to 5mm, packaged and sealed to obtain the yak feed.

[0015] Examples 2-4 The difference from Example 1 is that the volume ratio of the compound lactic acid bacteria agent is shown in Table 1 below.

[0016] Table 1. Volume ratio of compound lactic acid bacteria inoculants Lactobacillus plantarum Lactobacillus bruneri Lactobacillus fermentation Pediococcus pentosaceus Example 2 2 2 2 1 Example 3 4 3 2 1 Example 4 4 2 2 2

[0017] Comparative Examples 1-4 Unlike Example 1, the volume ratio of the compound lactic acid bacteria agent is different, as shown in Table 2 below.

[0018] Table 2 Volume ratio of compound lactic acid bacteria inoculant Lactobacillus plantarum Lactobacillus bruneri Lactobacillus fermentation Pediococcus pentosaceus Comparative Example 1 4 0 2 1 Comparative Example 2 4 1 2 0 Comparative Example 3 3 1 0 1 Comparative Example 4 4 4 0 0

[0019] Experimental Example 1: Determination of Nutrient Components in Fermented Feed The nutritional components of the yak feed prepared in each embodiment and each comparative example, including crude protein content, cellulose content, cellulose decomposition rate, and short-chain fatty acid content, were measured, and the results are shown in Table 3.

[0020] The methods used to detect the above indicators are as follows: Crude protein: Kjeldahl nitrogen determination method (GB / T 6432-2018); Cellulose degradation rate: Increment of reducing sugars determined by DNS method; Tannin content: Performed according to GB / T 27985-2011; Short-chain fatty acids: gas chromatography (total of acetic acid, propionic acid and butyric acid).

[0021] Table 3. Nutrient content of fermented feed Crude protein (%) Cellulose degradation rate (%) Tannins (mg / kg) Short-chain fatty acids (mg / g) Example 1 24.8 72.1 11.2 23.0 Example 2 22.5 68.6 13.5 21.4 Example 3 23.3 73.3 10.5 24.5 Example 4 25.2 72.4 10.8 24.2 Comparative Example 1 18.2 43.5 22.5 17.6 Comparative Example 2 17.4 40.2 18.6 16.9 Comparative Example 3 16.8 34.1 25.3 16.5 Comparative Example 4 15.1 40.3 32.4 19.4

[0022] The experimental results in Table 3 show that the fermented feed prepared from raw materials such as highland barley lees fermented according to the inoculation amount of compound lactic acid bacteria agent in Example 1 has high evaluation in terms of acidity, aroma and palatability. At the same time, the feed has almost no bitter taste and high nutritional value, with a crude protein content as high as 24.8%.

[0023] Example 2 reduced the inoculum amount of Lactobacillus plantarum compared to Example 1. The crude protein content and short-chain fatty acid content in the fermented feed were reduced. In addition, the degradation rate of cellulose was also reduced. The nutritional value of the fermented feed was lower than that of Example 1.

[0024] In Example 3, the content of Lactobacillus buchneri was increased. Although the cellulose degradation rate and short-chain fatty acid content in the obtained fermented feed were increased, the crude protein content did not increase significantly, but decreased slightly. Moreover, the feed had a strong sour taste and a slightly pungent odor, and its palatability was slightly worse than that of the fermented feed in Example 1.

[0025] In Example 4, the inoculum amount of Pediococcus pentosaceus was increased compared to Example 1. The degradation rate of cellulose was not significantly different from that in Example 1, but the protein content and short-chain fatty acid content were slightly increased.

[0026] Comparative Examples 1-4 omitted one or two strains of *Lactobacillus bruneri*, *Pediococcus pentosaceus*, and *Lactobacillus fermentum* from Example 1, and the amounts of other strains were slightly adjusted. The fermentation results showed a significant reduction in crude protein, short-chain fatty acid content, and cellulose degradation rate in the obtained fermented feed. Simultaneously, the fermented feed had a higher crude fiber content and improved chewiness. All the obtained feeds had a slightly bitter taste, indicating reduced palatability. This may be due to the reduced number of strains in the compound lactic acid bacteria agent, leading to a decreased degree of decomposition of bitter components such as tannins during fermentation, resulting in a slightly bitter taste in the fermented feed.

[0027] Experimental Example 2: Verification of the Antioxidant Effect of Fermented Feed Small amounts of fermented feed samples obtained from each example and comparative example were taken, and the content of the antioxidant component glutathione (GSH) in each fermented feed was determined by ultraviolet spectrophotometry. The HPPH free radical scavenging effect of each fermented feed was further verified to verify the antioxidant effect of each feed. The experimental results are shown in Table 4.

[0028] Table 4 Antioxidant effects of fermented feed GSH content (mg / g) DPPH free radical scavenging rate (%) Example 1 19.4 73.5 Example 2 18.1 64.2 Example 3 19.6 74.3 Example 4 19.8 72.1 Comparative Example 1 16.3 57.6 Comparative Example 2 15.8 54.3 Comparative Example 3 16.0 52.8 Comparative Example 4 16.9 55.7

[0029] Table 4 shows that the fermented feeds obtained by fermentation with different ratios of compound lactic acid bacteria agents all contained the antioxidant component glutathione. Among them, the fermented feeds obtained by the processes in Examples 1 to 4 had GSH content in the range of 18.1 mg / g to 19.8 mg / g, and had better antioxidant effect, at about 64.2% to 74.3%. This is of great significance for improving the immunity of yaks and reducing the disease rate of yaks during feeding.

[0030] The fermented feeds prepared in Comparative Examples 1-4 not only had lower GSH content, but also lower overall DPPH free radical scavenging rate. The reason for this phenomenon may be that when using compound lactic acid bacteria to ferment raw materials such as highland barley distillers' grains, on the one hand, various lactic acid bacteria secrete a variety of different active ingredients during fermentation, including small molecule components such as antimicrobial peptides, which exhibit certain antioxidant effects; on the other hand, the compound lactic acid bacteria agent decomposes the raw materials more thoroughly and fully, exposing the active groups, thereby greatly improving the overall antioxidant effect of the feed.

[0031] Experimental Example 3: Evaluation of the production performance of fermented feeds in each example and comparative example The experiment selected healthy male yaks around 2 years old with similar physical conditions and randomly divided them into 8 groups of 5 yaks each.

[0032] Fermented feeds from Examples 1-4 and Comparative Examples 1-4 were added to the daily diet of each group of yaks for a 60-day feeding trial. The weight gain rate and morbidity rate of the yaks were then recorded, and the results are shown in Table 5.

[0033] Table 5. Effects of fermented feed on weight gain and disease incidence in yaks. Average daily weight gain (kg / d) Average daily feed intake (kg / d) Number of infected heads (heads) Example 1 1.52 8.73 0 Example 2 1.48 8.69 1 Example 3 1.42 8.50 0 Example 4 1.37 7.66 0 Comparative Example 1 1.25 7.37 2 Comparative Example 2 1.10 7.25 2 Comparative Example 3 0.96 7.06 3 Comparative Example 4 0.82 6.44 2

[0034] Table 5 shows that the fermented feed prepared by fermenting highland barley lees with the compound lactic acid bacteria agent prepared in Examples 1-4 of this invention can reduce the disease rate of yaks in daily feeding, and the daily weight gain of yaks is within 1.37kg~1.52kg, and the daily feed intake of yaks is above 8 / 5kg. Only the fermented feed in Example 4 has a lower feed intake, which may be due to the slightly poor taste of the feed. In addition, the feed intake of the fermented feed in each comparative ratio is below 8kg, and the weight gain of yaks is also reduced. The reason for the above phenomena may be that the yield of compound lactic acid bacteria agent during fermentation process varies greatly, resulting in large differences in the chewiness, palatability, and aroma of fermented feed, which leads to a decrease in the feed intake of yaks.

[0035] In addition, after adding fermented feed in the above embodiments, the number of sick yaks during the feeding process was significantly reduced. Its excellent antioxidant effect and the active factors produced by probiotic fermentation are beneficial to improving the immunity and disease resistance of yaks.

Claims

1. A compound lactic acid bacteria agent for the fermentation of highland barley lees to produce yak feed, characterized in that, The compound lactic acid bacteria agent includes Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus fermentum, and Pediococcus pentosacchari. The compound lactic acid bacteria agent is prepared by first culturing each strain into a bacterial solution, and then mixing the bacterial solutions of Lactobacillus plantarum, Lactobacillus brucelli, Lactobacillus fermentum, and Pediococcus pentosacchari in a volume ratio of 2~4:1~3:1~3:0.5~2. In the compound lactic acid bacteria inoculant, the viable count of each lactic acid bacteria solution is 1.0 × 10⁻⁶. 9 cfu / mL ~1.0×10 11 cfu / mL.

2. The compound lactic acid bacteria agent as described in claim 1, characterized in that, The volume ratio of *Lactobacillus plantarum*: *Lactobacillus bruneri*: *Lactobacillus fermentum*: *Pediococcus pentosus* is 3~4:2:2:

1.

3. The method for preparing the compound lactic acid bacteria agent according to any one of claims 1 to 2, characterized in that, The steps include the following: S1 strain activation: Pick single colonies from the freeze-dried tubes and inoculate each single colony into MRS liquid medium, and incubate at 25~45℃ for 12~24h. S2 expansion culture: Inoculate the activated bacterial solution from S1 into the expansion culture medium at an inoculum rate of 0.5%~2% and expand the culture until the bacterial solution OD reaches the target value. 600 When the pH reaches 1.8–2.0, centrifuge, collect the bacterial sludge, wash 2–3 times with sterile physiological saline, and resuspend until the viable cell count in the bacterial culture reaches 1.0 × 10⁻⁶. 9 cfu / mL ~1.0×10 11 cfu / mL; S3 bacterial solution compounding: Mix the bacterial solutions of Lactobacillus plantarum, Lactobacillus bruneri, Lactobacillus fermentum and Pediococcus pentosaceus in a volume ratio of 3~4:2:2:1 to obtain the compound lactic acid bacteria inoculum.

4. The application of the compound lactic acid bacteria agent as described in any one of claims 1 to 2 in the preparation of yak feed from fermented highland barley lees.

5. The application as described in claim 4, characterized in that, The application includes the following steps: (1) Take barley lees, add alfalfa powder, barley bran, rapeseed cake, beet pulp, calcium carbonate and brown sugar to it, stir evenly, add water during stirring to adjust the moisture content of the material to 40%~60%, and pre-wet at room temperature for 12~24 hours; By weight, the ingredients are: 60-80 parts highland barley lees, 10-20 parts alfalfa hay powder, 5-15 parts highland barley bran, 5-10 parts rapeseed cake, 1-5 parts beet pulp, 0.5-1.5 parts calcium carbonate, and 1-2 parts brown sugar. (2) Spray the compound lactic acid bacteria agent evenly into the material prepared in (1) at an inoculation rate of 2%~8% (v / w) and stir thoroughly to mix evenly; (3) After inoculation, the material is put into a sealed fermentation tank, the air is exhausted, and fermentation is carried out at 20~28℃ for 2~3 days. During the fermentation process, the pH value is monitored and dipotassium hydrogen phosphate is added to maintain the pH at 5.5~6.

0. (4) After fermentation, the material is transferred to a drying equipment and dried at 40~45℃ until the moisture content of the material is ≤12%. After drying, it is crushed to 4~5mm, packaged and sealed to obtain the yak feed.