A method for producing single-cell protein compound feed using Napier grass silage

Abstract

This invention belongs to the fields of agricultural science and animal feed production, and discloses a method for producing single-cell protein compound feed using Napier grass silage, aiming to solve the technical problem of low crude protein content in Napier grass silage. This invention introduces *Bacillus tekirae*, which works synergistically with *Lactobacillus plantarum* and other bacteria to optimize the formulation and process parameters for producing single-cell protein compound feed from Guimu No. 1 and Wangcao silage, respectively. The true protein content of Guimu No. 1 is increased by 178.6%; the true protein content of Wangcao is increased by 84.3%. This invention can increase the protein content of forage, reduce feed and animal husbandry costs, promote the resource utilization of forage, and is of great significance for building a sustainable agricultural model.

Description

Technical Field

[0001] This invention belongs to the fields of agricultural science and animal feed production, specifically relating to a method for producing single-cell protein compound feed using Napier grass silage. Background Technology

[0002] Napier grasses are dominant cultivated forage grasses in tropical and subtropical regions of my country. Among them, the Guimu No. 1 hybrid Napier grass and Wang grass have seen their application in livestock production expand continuously in recent years due to their high biomass and wide adaptability, becoming an important source of roughage for ruminants. However, the crude protein content of Guimu No. 1 and Wang grass is generally low, making it difficult to meet the nutritional needs of livestock and poultry at different growth stages. This necessitates the addition of protein feed to balance the daily ration, leading to increased breeding costs and hindering the development of the livestock and poultry farming industry.

[0003] Single-cell protein (SCP), as a novel high-quality protein raw material, has gained widespread recognition for its application potential in animal feed due to its high protein content, balanced amino acid composition, and short production cycle. Currently, microbial fermentation technology is the mainstream technical route for SCP production. Utilizing the diverse metabolic networks of microorganisms, low-cost agricultural waste (such as straw and bran) and industrial organic wastewater can be used as fermentation substrates for resource transformation. Simultaneously, relying on the rapid proliferation characteristics of microorganisms, inexpensive carbon sources can be efficiently converted into SCP without the need for filtration, centrifugation, or concentration processes. This promotes waste reduction and resource utilization, yielding high-protein SCP compound feed, alleviating the supply and demand imbalance of high-quality protein feed, and is of great significance for optimizing ecosystem material cycling and building a sustainable agricultural development model. Among the microorganisms used in SCP compound feed production, yeast has become one of the core microbial groups in the current industrial production of SCP compound feed due to its flexible metabolic regulation, high safety, and stable protein synthesis efficiency.

[0004] Silage is a key technology for the resource utilization and long-term storage of forage. Anaerobic fermentation by microorganisms can lower the pH value of forage, inhibit the growth of putrefactive microorganisms, and thus improve palatability, digestibility, and shelf life, ensuring stable feeding for ruminants year-round. Notably, some yeast strains possess both sugar metabolism and lactic acid conversion capabilities, allowing them to synthesize their own proteins using sugar substrates in the silage system and lactic acid produced by lactic acid bacteria fermentation. This provides a technological possibility for in-situ production of single-cell protein compound feed coupled with silage. However, it should be noted that the forage silage process involves complex microbial community interactions, and the fermentation products are diverse. Some metabolites (such as excess organic acids and phenolic substances) can inhibit yeast growth and protein synthesis, preventing a breakthrough in the efficient production of single-cell protein compound feed within the silage system. Therefore, developing a method for efficiently producing single-cell protein compound feed using forage silage is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing single-cell protein compound feed using Napier grass silage. By optimizing the additive combination, the fermentation quality of Guimu No. 1 and Wangcao silage is improved, and these silages are further utilized to efficiently produce single-cell protein compound feed.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for a silage mixture for producing single-cell protein compound feed, wherein the mixture comprises, by mass percentage: 82.4%–90.34% silage hay powder, 9%–11% compound nutritional supplement, 0.5%–6% ammonium chloride, 0.04%–0.2% magnesium chloride, 0.04%–0.2% potassium chloride, and 0.04%–0.2% calcium chloride. The compound nutritional supplement is wheat bran, hornwort, or a mixture of wheat bran and hornwort, wherein the mass ratio of wheat bran to hornwort in the mixture is 3:0.1–0.1:3; preferably 3:1–1:3; more preferably 2:1–1:2.

[0008] Preferably, by mass percentage, the mixture comprises the following components: 84.55%–90.34% silage hay powder, 9%–10% compound nutritional supplement, 0.5%–5% ammonium chloride, 0.04%–0.15% magnesium chloride, 0.04%–0.15% potassium chloride, and 0.04%–0.15% calcium chloride. More preferably, by mass percentage, the mixture comprises the following components: 85.70%–90.34% silage hay powder, 9%–10% compound nutritional supplement, 0.5%–4% ammonium chloride, 0.04%–0.10% magnesium chloride, 0.04%–0.10% potassium chloride, and 0.04%–0.10% calcium chloride.

[0009] Furthermore, the silage hay powder is prepared by ensiling and fermenting Napier grass with additives, followed by drying and pulverizing; the additives are Bacillus tekirae, Lactobacillus plantarum, or a combination of Bacillus tekirae and Lactobacillus plantarum.

[0010] The aforementioned Napier grass forage is either Guimu No. 1 or Wangcao.

[0011] The Bacillus tequilensis strain described was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 3, 2021, with accession number CGMCC NO. 23715 (Patent No.: ZL202111444631.9), and the inoculum size was 1×10⁻⁶. 6 CFU / g FM or higher.

[0012] The *Lactobacillus plantarum* used was a commercially available product (*Lactobacillus plantarum MTD-1; EcosylProducts Ltd, Stokesley, North Yorkshire, UK, purchased from the market), and the inoculation amount was 1×10⁶. 6 CFU / g FM or higher.

[0013] Furthermore, the additive also contains urea. The amount of urea added is 0.5% to 2% (w / w).

[0014] Furthermore, the fermentation conditions are silage fermentation at 10~28℃ for 7~50 days;

[0015] Secondly, the present invention claims protection for a method for producing single-cell protein compound feed using the above-mentioned silage mixture, comprising the following steps:

[0016] (1) Weigh the silage mixture according to the formula ratio, add water to adjust the water-to-material ratio to 2~3:1, and sterilize it;

[0017] (2) Inoculate with Candida utilis and ferment at 5-45℃ for 24-96 hours;

[0018] (3) After fermentation, the mixture is dried and pulverized to obtain a single-cell protein compound feed.

[0019] The *Candida utilis* strain mentioned is a commercially available product with a viable count of 3.0 × 10⁻⁶ cells. 9 cfu / g.

[0020] Furthermore, the inoculation amount of Candida albicans is 5% to 20% (w / w).

[0021] Furthermore, the fermentation conditions are 30°C for 48–96 hours.

[0022] In a specific embodiment of the present invention, the method for producing single-cell protein compound feed includes the following steps:

[0023] (I) The silage is fermented by Lactobacillus plantarum or Bacillus tekirae alone, or by a combination of Lactobacillus plantarum, Bacillus tekirae, and urea (1% added, w / w) fermented to produce Guimu No. 1 or Wangcao, with an inoculum level of 1×10⁻⁶. 6 CFU / g FM or higher, silage fermented at room temperature (10~28℃) for 7~50 days (preferably 7~42 days), then dried at 80℃ and pulverized into silage hay powder through a 40-mesh screen.

[0024] (II) Weigh the silage mixture according to the formula ratio, add water, the water-to-material mass ratio is 2~3:1, seal with breathable sealing film and then sterilize.

[0025] (III) Inoculate the seed culture of Candida utilis at a rate of 5%–20% (w / w) into the sterilized silage mixture, mix well, and incubate in a fermentation incubator at 30 ℃ for 48–96 hours. After fermentation, place the samples before and after fermentation in an 80 ℃ drying oven to constant weight, and then pulverize to obtain single-cell protein compound feed.

[0026] As a specific embodiment, the optimal conditions for producing single-cell protein compound feed using Guimu No. 1 silage mixture treated with Lactobacillus plantarum alone, and the mass percentage of each component in the silage mixture, are as follows: Guimu No. 1 silage hay powder 88.00%, Elodea 9.78%, ammonium chloride 1.930%, magnesium chloride 0.098%, potassium chloride 0.098%, calcium chloride 0.098%, totaling approximately 100% (for the composition formulation, the sum of the percentages of each component should theoretically be 100%, but minor deviations due to rounding (usually within ±0.1%) do not affect the feasibility of the technical solution), water-to-material ratio 2:1, sealing with sealing film, high-pressure steam sterilization at 121 ℃ for 15 min, incubation at 30 ℃ for 72 h, Candida inoculation amount 15% (w / w), and true protein content increased by 178.6%.

[0027] The optimal conditions for producing single-cell protein compound feed using a mixture of *Lactobacillus plantarum*, *Bacillus tekirae*, and urea-treated *Wangcao* silage and the mass percentages of each component in the silage mixture are as follows: *Wangcao* silage hay powder 88.86%, wheat bran and *Ceratophyllum demersum* complex 9.87% (dry matter ratio of wheat bran to *Ceratophyllum demersum* 2:1), ammonium chloride 0.974%, magnesium chloride 0.0987%, potassium chloride 0.0987%, calcium chloride 0.0987%, totaling approximately 100% (theoretically, the sum of the percentages of each component should be 100% for the compound formulation; minor deviations due to rounding (usually within ±0.1%) do not affect the feasibility of the technical solution), water-to-material ratio 2:1, sealing with sealing film, autoclaving at 121 ℃ for 15 min, incubation at 30 ℃ for 96 h, *Candida* inoculation 10%, and true protein content increased by 84.3%.

[0028] Thirdly, this invention claims protection for single-cell protein compound feed produced by the above-described method. This single-cell protein compound feed is a protein widely used in animal feed, containing abundant protein and inorganic salts.

[0029] Fourthly, the present invention claims protection for the use of a combined additive in the preparation of the aforementioned silage mixture, said combined additive comprising Bacillus tequilensis QH1 (accession number CGMCC NO. 23715) and Lactobacillus plantarum. Further, said combined additive also comprises urea.

[0030] Fifthly, the present invention claims protection for the application of a combined additive in the optimization of fermentation process for single-cell protein compound feed, the combined additive comprising Bacillus equilensis QH1 with accession number CGMCC NO.23715 and Lactobacillus plantarum, used to optimize the culture substrate and conditions in the fermentation process with the growth rate of true protein content as an indicator.

[0031] Bacillus tequilensis QH1, a functional probiotic, produces various bioactive substances during its metabolism, including cellulase (such as cellulase and hemicellulase), α-tocopherol, and antimicrobial peptides. Bacillus tequilensis can improve nutrient accessibility by degrading lignocellulose in forage. This invention demonstrates that the synergistic effect of Bacillus tequilensis with common silage additives (such as lactic acid bacteria, enzymes, and urea) is expected to improve the microecological environment of silage systems, alleviate the inhibition of yeast by harmful metabolites during fermentation, and provide a new technological breakthrough for silage production coupled with SCP (silage processing).

[0032] This invention addresses the problem of low protein content in Napier grass forage by optimizing the combination of Bacillus tektii additives to improve silage quality and promote the production of single-cell protein compound feed. Results show that adding Lactobacillus plantarum alone and in combination with Bacillus tektii improves the fermentation quality of Guimu No. 1 and Wangcao silage. The optimal conditions for producing single-cell protein compound feed from Guimu No. 1 silage mixture treated with Lactobacillus plantarum alone, and the mass ratio of each component in the silage mixture, are as follows: Guimu No. 1 silage hay powder 88.00%, Elodea spp. 9.78%, ammonium chloride 1.930%, magnesium chloride 0.098%, potassium chloride 0.098%, calcium chloride 0.098%, totaling approximately 100%, water-to-material ratio 2:1, sealed with sealing film, autoclaved at 121 ℃ for 15 min, incubated at 30 ℃ for 72 h, Candida albicans inoculation 15% (w / w), and true protein content increased by 178.6%. The optimal conditions for producing single-cell protein compound feed from a mixture of *Lactobacillus plantarum*, *Bacillus tekirae*, and urea (1%, w / w) treated with *Lactobacillus plantarum*, *Bacillus tekirae*, and urea (1%, w / w) were as follows: *Lactobacillus plantarum* hay powder 88.86%, wheat bran and *Ceratophyllum demersum* complex 9.87% (wheat bran and *Ceratophyllum demersum* dry matter mass ratio 2:1), ammonium chloride 0.974%, magnesium chloride 0.0987%, potassium chloride 0.0987%, calcium chloride 0.0987%, totaling approximately 100%, water-to-material ratio 2:1, sealed with sealing film, autoclaved at 121 °C for 15 min, incubated at 30 °C for 96 h, *Candida* inoculation 10%, and true protein content increased by 84.3%.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Using high-quality fermented Napier grass silage to produce single-cell protein compound feed is low-cost, safe, reliable and easy to apply.

[0035] (2) Using Napier grass silage to produce single-cell protein compound feed has a stable supply of raw materials, a long shelf life, and high nutritional value.

[0036] (3) The present invention can effectively increase the true protein content of silage mixture and is rich in inorganic salts required by animals. Attached Figure Description

[0037] Figure 1 The effect of additives on the crude protein and fiber content of Guimu No. 1 silage after 42 days of fermentation; different lowercase letters on the column represent significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea.

[0038] Figure 2 Effects of additives on crude protein and fiber content of Wangcao silage fermented for 42 days; different lowercase letters on the column represent significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea.

[0039] Figure 3 The effects of additives and incubation time on the production of single-cell protein compound feed from Guimu No. 1 silage were investigated. Different lowercase letters on the columns represent significant differences (P < 0.05). CK: Control; B: QH1 bacteria; N: 1% urea; BN: 1% urea + QH1 bacteria; Y: Lactobacillus plantarum; BY: QH1 bacteria + Lactobacillus plantarum; BYN: QH1 bacteria + Lactobacillus plantarum + 1% urea; A: Additives; T: Silage time; A×T: Interaction between additives and silage time.

[0040] Figure 4 The effects of additives and incubation time on the production of single-cell protein compound feed from *Lactobacillus plantarum* silage were investigated. Different lowercase letters on the columns represent significant differences (P < 0.05). CK: Control; B: QH1 bacteria; N: 1% urea; BN: 1% urea + QH1 bacteria; Y: *Lactobacillus plantarum*; BY: QH1 bacteria + *Lactobacillus plantarum*; BYN: QH1 bacteria + *Lactobacillus plantarum* + 1% urea; A: Additives; T: Silage time; A×T: Interaction between additives and silage time.

[0041] Figure 5 The effect of additives on the increase rate of true protein content in four types of silage; different lowercase letters on the columns represent significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] Example 1: Effects of QH1 and its combination with additives on the fermentation quality of Guimu No. 1 and Wangcao silage

[0044] 1. Materials and Methods

[0045] 1.1 Source of materials

[0046] The Guimu No. 1 and Wangcao varieties used in the experiment were obtained from the Baima Base of Nanjing Agricultural University and are well-known forage varieties of the genus Napier grass. Whole plants of Guimu No. 1 and Wangcao were harvested on October 25, 2022, for silage. The silage material was chopped into small pieces using a laboratory hay cutter for easy compaction in the silo, and then mixed thoroughly before use. Urea and Lactobacillus plantarum MTD-1 (Ecosyl Products Ltd, Stokesley, North Yorkshire, UK) used in the experiment were purchased from the market. Bacillus tequilensis QH1 (QH1, deposited on November 3, 2021, at the China General Microbiological Culture Collection Center, accession number CGMCC NO. 23715; patent number: ZL202111444631.9) was obtained from our laboratory.

[0047] 1.2 Experimental Design

[0048] The experiment was designed as a two-factor randomized block design: 7 treatment groups [control group (CK); QH1 bacteria (QH1), inoculum size 1×10⁻⁶]. 6 cfu / g FM; 1% (w / w) urea (N); QH1 bacteria + 1% urea (BN); Lactobacillus plantarum (Y), inoculum size 1×10 6 cfu / g FM; QH1 bacteria + Lactobacillus plantarum (BY), inoculation amount of each is 1×106 cfu / g FM; QH1 bacteria + Lactobacillus plantarum + 1% urea (BYN)], inoculation amount of each is 1×10 6 cfu / g FM and silage time (7, 21 and 42 d), with 4 replicates for each treatment.

[0049] 1.3 Test Methods

[0050] 1.3.1 Silage preparation

[0051] Polyethylene laboratory silage bags were used to fill the silage material. Each bag contained 120 g of Guimu No. 1 or Wangcao silage. The appropriate amount of additives was added according to the experimental design, and the mixture was thoroughly mixed. The bags were then vacuum-sealed using a laboratory vacuum packaging machine and stored in the dark, away from light, at room temperature (10–28 °C). Samples from four silage bags were randomly selected at 7, 21, and 42 days after ensiling and analyzed to determine the fermentation and nutritional quality of the silage.

[0052] 1.3.2 Sample Preparation

[0053] After opening the silage bags, thoroughly mix the silage sample. Weigh 25 g of Guimu No. 1 or Wangcao using the "quartering method" and pour it into a 100 mL glass conical flask. Add 75 mL of distilled water and place the flask in a 4 ℃ refrigerator for 24 h of extraction. Filter the extract through double-layer gauze and qualitative filter paper to obtain the silage sample extract filtrate, which is then frozen at -20 ℃ for testing. The extract filtrate is used to determine the pH value, organic acid content, and ammonia nitrogen content of the silage sample. Separately, take 10 g of the sample and place it in a 100 mL conical flask. Add 90 mL of 0.85% (m / v, g / 100 mL) sterile physiological saline solution and place the flask on a shaker at 120 rpm for 2 hours. The resulting bacterial solution is used to determine the number of aerobic bacteria, yeast, and lactic acid bacteria. Approximately 50 g of sample was weighed and dried in a 65 ℃ oven until constant weight was achieved, and the dry matter (DM) content was determined. After drying, the sample was weighed again, pulverized into grass powder, and passed through a 1 mm sieve for the determination of crude protein (CP), water-soluble carbohydrates (WSC), neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) contents. 10 g of sample was weighed into a 50 mL centrifuge tube, sealed with gauze, and frozen at -80 ℃ for subsequent determination of α-tocopherol content.

[0054] 1.4 Measurement Indicators and Analytical Methods

[0055] 1.4.1 pH value

[0056] The pH value of silage extract was determined using a pH 211 precision pH meter (HANNA, Italy) (Liu Qinhua et al., 2009).

[0057] 1.4.2 Organic acids

[0058] The content of organic acids was determined by high performance liquid chromatography [Yang L, Yuan X, Li J, et al. Dynamics of microbial community and fermentation quality during ensiling ofsterile and nonsterile alfalfa with or without Lactobacillus plantaruminoculant[J]. Bioresource Technology, 2019, 275: 280-287.].

[0059] 1.4.3 Ammonia nitrogen

[0060] The ammonia nitrogen content was determined by the phenol-sodium hypochlorite colorimetric method [Broderick GA, Kang J H. Automated simultaneous determination of ammonia and total amino acids inruminal fluid and in vitro media[J]. Journal of Dairy Science, 1980, 63(1):64-75.] and expressed as total nitrogen basis (g / kg TN).

[0061] 1.4.4 Crude protein and total nitrogen

[0062] The total nitrogen content in silage was determined using a Kjeltec™ 2300 fully automatic Kjeldahl nitrogen analyzer (Foss, Denmark). The crude protein content was calculated by multiplying the total nitrogen content by 6.25 [Yang Sheng. Feed Analysis and Feed Quality Testing Technology [M]. Feed Analysis and Feed Quality Testing Technology, 1993.].

[0063] 1.4.5 Water-soluble carbohydrates

[0064] The anthrone-sulfuric acid colorimetric method was used to determine the water-soluble carbohydrate content in silage [Yan YH, Li J L, Guo XS, et al. A study on fermentation quality of Italian ryegrass and soybean straw mixed silage[J]. Acta Prataculturae Sinica, 2014, 23(4): 94-99.].

[0065] 1.4.6 Neutral detergent fibers, acid detergent fibers and acid detergent lignin

[0066] The content of neutral detergent fiber, acid detergent fiber and acid detergent lignin in silage was determined using the method of Van Soest et al. [Wang SR, Yuan XJ, Dong ZH, et al. Isolating and evaluating lactic acid bacteria strains for effectiveness on silage quality at low temperatures on the Tibetan Plateau[J]. Animal Science Journal, 2017, 88(11):1722-1729.].

[0067] 1.4.7 Buffer Energy

[0068] Determination of buffering energy of forage by hydrochloric acid-sodium hydroxide titration [Chen L, Guo G, Yu CQ, et al. The effects of replacement of whole-plant corn with oat and common vetch on the fermentation quality, chemical composition and aerobic stability of total mixed ration silage in Tibet[J]. Animal Science Journal, 2015, 86(1): 69-76.].

[0069] 1.4.8 Microbial Quantity Determination

[0070] Microbial counting was performed using the plate culture method. Aerobic bacteria were cultured on nutrient agar (NA) medium at 37 °C for 24 h under aerobic conditions, and the colony count was then recorded. Yeast was cultured on potato dextrose agar (PDA) medium at 30 °C for 48 h under aerobic conditions, and the colony count was then recorded. Lactic acid bacteria (LAB) were cultured on de Man-Rogosa-Sharpe agar medium at 37 °C for 48 h under anaerobic conditions, and the colony count was then recorded.

[0071] 1.5 Data Statistical Analysis

[0072] The experimental data were statistically analyzed using IBM SPSS Statistics 22.0 software. Two-way ANOVA was used for the chemical and microbiological data of silage. Duncan's multiple comparisons were employed to analyze the differences between data means, with P < 0.05.

[0073] 2 Results and Analysis

[0074] 2.1 Chemical and microbial components of Guimu No. 1 and Wangcao Fresh Samples

[0075] As shown in Table 1, the dry matter content of fresh Guimu No. 1 was 221 g / kg FM, while that of fresh Wangcao was 216 g / kg FM. The pH values ​​of both Guimu No. 1 and Wangcao were relatively high, at 6.09 and 6.11 respectively, with buffering energies of 63.4 and 113.0 mEq / kg DM respectively. The crude protein and water-soluble carbohydrate contents of fresh Guimu No. 1 were 61.9 and 52.8 g / kg DM, respectively, while those of fresh Wangcao were 73.9 and 36.9 g / kg DM, respectively. The contents of neutral detergent fiber, acid detergent fiber, and acid detergent lignin before silage of Guimu No. 1 were 658, 406, and 58.6 g / kg DM, respectively, while those of Wangcao were 635, 400, and 60.8 g / kg DM, respectively. The number of lactic acid bacteria, aerobic bacteria, and yeast attached to the fresh sample of Guimu No. 1 were 6.42, 9.03, and 8.61 lg CFU / g FM, respectively, while the number of lactic acid bacteria, aerobic bacteria, and yeast attached to the fresh sample of Wangcao were 6.29, 9.21, and 8.72 lg CFU / g FM, respectively. The α-tocopherol content of the fresh samples of Guimu No. 1 and Wangcao was 7.40 and 11.6 mg / kg DM, respectively.

[0076] Table 1. Chemical and microbial components of Guimu No. 1 and Wangcao fresh samples.

[0077]

[0078] Note: DM, dry matter; FM, fresh matter; mEq, milligram equivalent; cfu, colony unit.

[0079] 2.2 Effects of additives and silage time on the fermentation quality of Guimu No. 1 and Wangcao silage

[0080] As shown in Table 2, additives significantly affected the pH, lactic acid, acetic acid, butyric acid, ethanol, ammonia nitrogen content, and LA / AA ratio of Guimu No. 1 silage (P < 0.05). Silage time significantly affected the pH, acetic acid, butyric acid, ammonia nitrogen content, and LA / AA ratio of Guimu No. 1 silage (P < 0.05). The interaction between additives and silage time significantly affected the lactic acid, acetic acid, butyric acid, ammonia nitrogen content, and pH of Guimu No. 1 silage (P < 0.05).

[0081] When different treatments included the silage time factor, the LA / AA ratio of Guimu No. 1 silage Y and BY groups was the highest, significantly higher than other treatment groups (P < 0.05); the LA / AA ratio of the BN group was the lowest, while the ethanol content was the highest.

[0082] Table 2. Effects of additives and silage time on the fermentation quality of Guimu No. 1 silage.

[0083]

[0084] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea; LA / AA, lactobacillus-to-ethyl ratio.

[0085] When the silage time covers different treatment factors, the LA / AA value of Guimu No. 1 silage generally shows a decreasing trend during the silage process.

[0086] When considering the interaction between different treatments and silage time, the pH value of Guimu No. 1 silage reached its highest point after 7 days of silage in the BN group and its lowest point after 42 days of silage in the Y group. Lactic acid content was highest in the Y group after 21 days of silage and lowest in the BN group after 21 days of silage. Acetic acid content was highest in the N group after 42 days of silage and lowest in the BY group after 7 days of silage. Butyric acid content was highest in the BN group after 21 days of silage. Ammonia nitrogen content was highest in the N group after 42 days of silage and lowest in the BY group after 7 days of silage.

[0087] As shown in Table 3, the interaction between additives and ensiling time significantly affected the pH, lactic acid, acetic acid, propionic acid, butyric acid, ammonia nitrogen content, and LA / AA ratio of *Cynanchum paniculatum* silage (P < 0.05). Additives significantly affected the pH, lactic acid, acetic acid, propionic acid, butyric acid, ethanol, ammonia nitrogen content, and LA / AA ratio of *Cynanchum paniculatum* silage (P < 0.05). Ensiling time significantly affected the pH, acetic acid, propionic acid, butyric acid, ethanol, ammonia nitrogen content, and LA / AA ratio of *Cynanchum paniculatum* silage (P < 0.05).

[0088] When different treatments included silage time, the ethanol content in the Wangcao silage BN group was significantly higher than that in the CK group (P < 0.05).

[0089] When the silage time covers different treatment factors, the ethanol content of whole-grain silage generally shows a decreasing trend with the silage time.

[0090] Considering the interaction between different treatments and silage time, the pH value of *Corydalis yanhusuo* silage was highest in groups N and BN after 7 days of silage, and lowest in group Y after 7 days of silage. Lactic acid content was highest in group BY after 42 days of silage. Acetic acid content was highest in group BYN after 42 days of silage, and lowest in group Y after 7 days of silage. Propionic acid content was highest in group CK after 7 days of silage. Butyric acid content was highest in group BYN after 42 days of silage. Changes in ammonia nitrogen content during silage reflected the fermentation quality of the silage. The study showed that ammonia nitrogen content reached its highest in group N after 42 days of silage, while it was lowest in group Y after 7 days of silage. The LA / AA ratio was highest in group Y after 7 days of silage.

[0091] 2.3 Effects of additives and silage time on the microbial composition of Guimu No. 1 and Wangcao silage

[0092] As shown in Table 4, the interaction between additives and silage time significantly affected the number of lactic acid bacteria, aerobic bacteria, and yeast in Guimu No. 1 silage (P < 0.05). Additives significantly affected the number of lactic acid bacteria, aerobic bacteria, and yeast in Guimu No. 1 silage (P < 0.05). Silage time significantly affected the number of aerobic bacteria and yeast in Guimu No. 1 silage (P < 0.05).

[0093] After considering the interaction between different treatments and silage time, it was found that the number of aerobic bacteria in Guimu No. 1 silage reached its highest level in the BN group after 42 days of silage, while it was the lowest in the B group after 42 days of silage. The number of yeasts was the highest in the BYN group after 7 days of silage. The number of lactic acid bacteria was also the highest in the BN group after 42 days of silage, and the lowest in the Y group after 42 days of silage.

[0094] As shown in Table 5, additives significantly affected the number of lactic acid bacteria, aerobic bacteria, and yeast in Wangcao silage (P < 0.05). Silage time significantly affected the number of lactic acid bacteria, aerobic bacteria, and yeast in Wangcao silage (P < 0.05). The interaction between additives and silage time significantly affected the number of lactic acid bacteria, aerobic bacteria, and yeast in Wangcao silage (P < 0.05).

[0095] Considering the interaction between different treatments and silage time, the number of aerobic bacteria in Wangcao silage was highest in group BN after 21 days of silage and lowest in group B after 42 days of silage. The number of yeasts was highest in group CK after 7 days of silage. The number of lactic acid bacteria was highest in group BN after 42 days of silage and lowest in group Y after 21 days of silage.

[0096] Table 3. Effects of additives and silage time on the fermentation quality of Wangcao silage.

[0097]

[0098] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea; LA / AA, lactobacillus-to-ethyl ratio.

[0099] 2.4 Effects of additives and silage time on the nutritional quality of Guimu No. 1 and Wangcao silage

[0100] As shown in Table 6, the interaction between additives and silage time had no significant effect on the DM and WSC of Guimu No. 1 silage (P > 0.05). Both additives and silage time significantly affected the DM and WSC of Guimu No. 1 silage (P < 0.05).

[0101] When different treatments included silage time, the DM of Guimu No. 1 silage N and BN groups was significantly lower than that of other treatment groups (P < 0.05); compared with the CK group, BN and BYN groups significantly reduced WSC (P < 0.05), while BY group significantly increased WSC (P < 0.05).

[0102] Table 4. Effects of additives and silage time on the microbial composition of Guimu No. 1 silage.

[0103]

[0104] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea.

[0105] When the silage time covered different treatment factors, the DM of Guimu No. 1 silage first decreased and then remained unchanged during the silage process, while the WSC first decreased and then increased during the silage process, reaching its lowest point at 21 days.

[0106] The effects of additives on the CP, NDF, ADF and ADL content of Guimu No. 1 silage on day 42 of fermentation are as follows: Figure 1 As shown, different additive treatments significantly affected the ADF, ADL, and CP content of Guimu No. 1 silage (P < 0.05). Compared with the CK group, the BN and BYN groups significantly increased the ADF and ADL content (P < 0.05) and significantly decreased the CP content (P < 0.05).

[0107] Table 5. Effects of additives and silage time on the microbial composition of *Cynanchum paniculatum* silage.

[0108]

[0109] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control; B, QH1 bacteria; N, 1% urea; BN, 1% urea + QH1 bacteria; Y, Lactobacillus plantarum; BY, QH1 bacteria + Lactobacillus plantarum; BYN, QH1 bacteria + Lactobacillus plantarum + 1% urea.

[0110] As shown in Table 7, both additives and silage time significantly affected the DM and WSC of *Cynanchum paniculatum* silage (P < 0.05). The interaction between additives and silage time had no significant effect on the DM and WSC of *Cynanchum paniculatum* silage (P > 0.05).

[0111] When different treatments included silage time, the DM of the N and BN groups of Wangcao silage was significantly lower than that of the CK group (P < 0.05), with the N group having the lowest DM; the WSC of the Y, BY and BYN groups was significantly higher than that of the CK group (P < 0.05), with the BY group having the highest WSC.

[0112] Table 6. Effects of additives and silage time on the dry matter and water-soluble carbohydrate content of Guimu No. 1 silage.

[0113]

[0114] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control, no additive; B, QH1 strain; N, 1% urea; BN, 1% urea and QH1 strain; Y, Lactobacillus plantarum; BY, QH1 strain and Lactobacillus plantarum; BYN, QH1 strain and Lactobacillus plantarum and 1% urea; DM, dry matter; WSC, water-soluble carbohydrates.

[0115] Under different ensiling times covering various treatment factors, the dry matter (DM) content of Wangcao silage first decreased and then stabilized with ensiling time, while the water-soluble carbohydrate (WSC) content remained unchanged and then increased during ensiling.

[0116] like Figure 2 As shown, the study found that by adding specific additives, the contents of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) in Wangcao silage were significantly affected on day 42 of fermentation. Different additive treatments significantly affected the contents of NDF, ADF, ADL, and CP in Wangcao silage (P < 0.05). The CP contents of the N, BN, and BYN groups were significantly lower than those of the CK group (P < 0.05), while the CP contents of the Y and BY groups were significantly higher than those of the CK group (P < 0.05). The BY group had the lowest NDF and ADF contents, significantly lower than those of the CK group (P < 0.05). The BN group had the highest contents of NDF, ADF, and ADL, with significantly higher ADF and ADL contents compared to the CK group (P < 0.05).

[0117] Table 7. Effects of additives and silage time on the nutritional quality of *Cynanchum paniculatum* silage.

[0118]

[0119] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05); CK, control, no additive; B, QH1 strain; N, 1% urea; BN, 1% urea and QH1 strain; Y, Lactobacillus plantarum strain; BY, QH1 strain and Lactobacillus plantarum; BYN, QH1 strain and Lactobacillus plantarum and 1% urea; DM, dry matter; WSC, water-soluble carbohydrates.

[0120] In conclusion, different additives have a significant impact on the fermentation quality and nutritional quality of Guimu No. 1 and Wangcao silage, and whether they can be used to produce single-cell protein compound feed requires further research.

[0121] Example 2: Production of single-cell protein compound feed from a mixture of Napier grass silage

[0122] 1. Materials and Methods

[0123] 1.1 Test Materials

[0124] Guimu No. 1 and Wangcao silage were obtained from the above silage experiments. The wheat bran used in the experiment was purchased from the Baima Town Farmers Market in Nanjing; the goldfish algae was taken from Yueya Lake in Nanjing; the yeast—Candida utilis Y5—was purchased from an online merchant (Junchang Biological Laboratory, viable count 3 billion / gram).

[0125] 1.2 Test Methods

[0126] 1.2.1 Research on the Production of Single-Cell Protein Compound Feed from Silage

[0127] Yeast seed culture: Yeast Y5 was activated and inoculated into 50 mL of yeast extract peptone (YPD) liquid medium under aseptic conditions in a clean bench and cultured at 30 ℃ for 24 h.

[0128] Preliminary test formulation and fermentation conditions: Weigh 2 g of substrate (1.8 g of hay powder from any type of Napier grass silage for 42 days, accounting for 88.85% of the total, and 0.2 g of wheat bran, accounting for 9.87% of the total). Based on the substrate weight, add other culture medium components (w / w): 1% ammonium chloride, accounting for 0.987% of the total, 0.1% magnesium chloride, accounting for 0.0987% of the total, 0.1% potassium chloride, accounting for 0.0987% of the total, and 0.1% calcium chloride, accounting for 0.0987% of the total) into a 100 mL Erlenmeyer flask. The water-to-substrate ratio is 2.5:1. Seal the flask with sealing film and autoclave at 121 ℃ for 15 min. Prepare several portions for later use. Yeast seed culture was inoculated into 100 mL sterile Erlenmeyer flasks at a 10% inoculation rate (w / w) using a pipette. After mixing, the flasks were incubated at 30 °C for 72 h. After fermentation, the samples before and after fermentation were dried in an 80 °C oven until constant weight, pulverized, and passed through a 1 mm sieve. The true protein content was then determined. Additives that improve the true protein content of single-cell protein compound feed in silage production were screened out.

[0129] 1.2.2 Optimization of key factors affecting the production of single-cell protein compound feed from Napier grass forage silage

[0130] Based on the preliminary experimental results, the silage from the Guimu No. 1 Y treatment group and the Wangcao BYN treatment group on day 42 was selected for optimization experiments on key factors affecting the production of single-cell protein compound feed. In the fermentation medium formula, waterweed (Ceratophyllum demersum, dry matter content 56.9 g / kg FM) partially replaced wheat bran on a dry matter basis, as shown in Table 8. Considering the high energy consumption of drying after fermentation, the water-to-material ratio was adjusted to 2:1.

[0131] An L16 (4+4) orthogonal design was used, with the increase in true protein content as the evaluation index. Four factors were considered during fermentation: the ratio of wheat bran to aquatic plants, the amount of ammonium chloride added, the culture time, and the inoculum quantity. Four levels were selected for orthogonal experiments to determine suitable fermentation conditions. Based on the orthogonal results, suitable condition combinations were determined. If the orthogonal experimental combination did not include a suitable condition combination, a validation experiment was conducted. The design of the response factors and levels is shown in Table 8.

[0132] 1.3 Measurement Indicators and Analytical Methods

[0133] The true protein content was determined using the trichloroacetic acid precipitation method: 0.5 g of sample was weighed and placed in a 100 mL Erlenmeyer flask, 50 mL of distilled water was added and the mixture was allowed to stand for 30 min; then 10 mL of 10% trichloroacetic acid (w / v) solution was added and the mixture was allowed to stand for 20 min; the liquid was filtered through neutral quantitative filter paper, and the filter residue was rinsed 2-3 times with 10% trichloroacetic acid solution; the filter paper and filter residue were dried together in an oven at 80 ℃ and then transferred to a Kjeldahl flask for protein determination using the Kjeldahl nitrogen determination method.

[0134] Table 8. Orthogonal Experiment Factors and Levels Design (Designed based on preliminary experimental results)

[0135]

[0136] 1.4 Data Statistical Analysis

[0137] After importing the experimental data into IBM SPSS Statistics 22.0, we performed a one-way ANOVA and applied Duncan's multiple comparison method to analyze the significance of the differences in the means between different groups. The significance level was set to P < 0.05.

[0138] 2 Results and Analysis

[0139] 2.1 Screening of Additives for Fermentation of Single-Cell Protein Compound Feed from a Mixture of Guimu No. 1 and Wangcao Silage

[0140] The effects of additives on the true protein content of Guimu No. 1 and Wangcao silage before and after fermentation are shown in the preliminary experimental results. Figure 3 and Figure 4 As shown in the figure. Compared with before fermentation, the true protein content of Guimu No. 1 and Wangcao silage group N increased significantly. The effect of additives on the increase rate of true protein content in Guimu No. 1 and Wangcao silage is shown in the figure. Figure 5 As shown. After 3 days of fermentation, the increase rate of true protein content in the Y and BY groups of Guimu No. 1 silage was significantly higher than that in the CK group (P < 0.05), with the Y group showing the highest increase rate of true protein content; the increase rate of true protein content in the BYN group of Wangcao silage was significantly higher than that in the CK group (P < 0.05).

[0141] In summary, the silage from the Guimu No. 1 Y treatment group and the Wangcao BYN treatment group on day 42 was selected for subsequent optimization experiments on key factors affecting the production of single-cell protein compound feed.

[0142] 2.2 Orthogonal Experiment Analysis

[0143] 2.2.1 Orthogonal Experimental Analysis of Guimu No. 1

[0144] The results of the orthogonal experiment are shown in Table 9. According to range analysis, the influence of each factor on the increase rate of true protein content can be ranked as follows: culture time > inoculum quantity > ammonium chloride addition > bran-aquatic plant ratio. The optimal experimental conditions were: bran-aquatic plant ratio 3:0, ammonium chloride addition 3.948%, culture time 72 h, and inoculum quantity 15% (w / w). Therefore, within the scope of this experimental design, the intuitively optimal combination is A1B3C3D3, and the theoretically optimal combination is A4B2C3D3, i.e., bran-aquatic plant ratio 0:3, ammonium chloride addition 1.974%, culture time 72 h, and inoculum quantity 15%.

[0145] When conducting orthogonal experimental design, the theoretically predicted optimal protein-producing combination A4B2C3D3 was not included. To verify this prediction, a specific validation experiment was performed. The results showed that after experimenting with the theoretically predicted optimal combination, the increase in true protein content reached 178.6%. This result not only validated the accuracy of the theoretical prediction but also indicated that this combination achieved a higher true protein content compared to the intuitively chosen optimal combination, fully meeting expectations.

[0146] Table 9. Orthogonal experimental conditions and results of Guimu No. 1 silage mixture

[0147]

[0148] 2.2.2 Analysis of Wangcao Orthogonal Experiment

[0149] The results of the orthogonal experiment are shown in Table 10. According to range analysis, the influence of each factor on the increase rate of true protein content can be ranked as follows: culture time > bran-to-water ratio > ammonium chloride concentration > inoculum quantity. The optimal experimental conditions were a bran-to-water ratio of 2:1, ammonium chloride addition of 3.948%, a culture time of 96 h, and an inoculum quantity of 5%. Therefore, within the scope of this experimental design, the intuitively optimal combination is A2B3C4D1, and the theoretically optimal combination is A2B1C4D2, i.e., a bran-to-water ratio of 2:1, ammonium chloride addition of 0.987%, a culture time of 96 h, and an inoculum quantity of 10%.

[0150] Since the theoretically optimal protein production combination is A2B1C4D2, which is not included in the orthogonal experimental treatment combination, a verification experiment was conducted. The results showed that the theoretically optimal combination A2B1C4D2 increased the true protein content by 84.3%, which is higher than the intuitively optimal combination and meets expectations.

[0151] Table 10. Orthogonal experimental conditions and results of Wangcao silage mixture.

[0152]

[0153] In summary, silage can be used to produce single-cell protein compound feed. The Y treatment group of Guimu No. 1 silage and the BYN treatment group of Wangcao silage showed the best results in producing single-cell protein compound feed. The optimal conditions for producing single-cell protein compound feed from Guimu No. 1 silage mixture treated with Lactobacillus plantarum alone and the mass ratio of each component of the silage mixture are as follows: Guimu No. 1 silage hay powder 88.00%, Elodea spp. 9.78%, ammonium chloride 1.930%, magnesium chloride 0.098%, potassium chloride 0.098%, calcium chloride 0.098%, totaling approximately 100%, water-to-material ratio 2:1, sealed with sealing film, autoclaved at 121 ℃ for 15 min, incubated at 30 ℃ for 72 h, Candida albicans inoculation amount 15% (w / w), and true protein content increased by 178.6%. The optimal conditions for producing single-cell protein compound feed from a mixture of *Lactobacillus plantarum*, *Bacillus tekirae*, and urea-treated *Wangcao* silage and the mass proportions of each component in the silage mixture are as follows: *Wangcao* silage hay powder 88.86%, wheat bran and *Ceratophyllum demersum* complex 9.87% (wheat bran and *Ceratophyllum demersum* dry matter mass ratio 2:1), ammonium chloride 0.974%, magnesium chloride 0.0987%, potassium chloride 0.0987%, calcium chloride 0.0987%, totaling approximately 100%, water-to-material ratio 2:1, sealed with sealing film, autoclaved at 121℃ for 15 min, incubated at 30℃ for 96 h, *Candida* inoculation 10%, and true protein content increased by 84.3%.

Claims

1. A silage mixture for producing single-cell protein compound feed, characterized in that, The mixture comprises the following components by mass percentage: 82.4%–90.34% silage hay powder, 9%–11% compound nutrient supplement, 0.5%–6% ammonium chloride, 0.04%–0.2% magnesium chloride, 0.04%–0.2% potassium chloride, and 0.04%–0.2% calcium chloride; the compound nutrient supplement is wheat bran, hornwort, or a mixture of wheat bran and hornwort, wherein the mass ratio of wheat bran to hornwort in the mixture is 3:0.1–0.1:3; preferably 3:1–1:3; more preferably 2:1–1:

2.

2. The silage mixture according to claim 1, characterized in that, The mixture comprises, by mass percentage: 84.55%–90.34% silage hay powder, 9%–10% compound nutrient supplement, 0.5%–5% ammonium chloride, 0.04%–0.15% magnesium chloride, 0.04%–0.15% potassium chloride, and 0.04%–0.15% calcium chloride; preferably, the mixture comprises, by mass percentage: 85.70%–90.34% silage hay powder, 9%–10% compound nutrient supplement, 0.5%–4% ammonium chloride, 0.04%–0.10% magnesium chloride, 0.04%–0.10% potassium chloride, and 0.04%–0.10% calcium chloride.

3. The silage mixture according to claim 1 or 2, characterized in that, The silage hay powder is made from Napier grass fodder that has been fermented with additives, then dried and pulverized; the additives are Bacillus tekirae, Lactobacillus plantarum, or a combination of Bacillus tekirae and Lactobacillus plantarum; the Napier grass fodder is Guimu No. 1 or Wangcao; the preservation number of Bacillus tekirae is CGMCC NO. 23715.

4. The silage mixture according to claim 3, characterized in that, The additive also contains urea, and the amount of urea added is 0.5% to 2% (w / w).

5. A method for producing single-cell protein compound feed using the silage mixture according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Weigh the silage mixture according to the formula ratio, add water to adjust the water-to-material ratio to 2~3:1, and sterilize it; (2) Inoculate with Candida albicans and ferment at 5-45℃ for 24-96 hours; (3) After fermentation, the mixture is dried and pulverized to obtain a single-cell protein compound feed.

6. The method according to claim 5, characterized in that, The inoculation amount of Candida albicans is 5% to 20%.

7. The method according to claim 5 or 6, characterized in that, The fermentation conditions are 30°C for 48–96 hours.

8. A single-cell protein compound feed produced by the method described in any one of claims 5-7.

9. The use of a combination additive in the preparation of the silage mixture according to any one of claims 1-4, characterized in that, The additive combination contains Bacillus tekirae and Lactobacillus plantarum.

10. The application according to claim 9, characterized in that, The additive combination also includes urea.

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