Pichia kudriavzevii SJTU-BEL-1 for producing single-cell protein and application of Pichia kudriavzevii SJTU-BEL-1

Abstract

The invention relates to the technical field of microorganisms, in particular to pichia kudriavzevii SJTU-BEL-1 capable of producing single-cell protein and application of the pichia kudriavzevii SJTU-BEL-1. The pichia kudriavzevii SJTU-BEL-1 provided by the invention is a strain obtained by performing primary screening, secondary screening and identification on supermarket over-ripe strawberry residues, and when the pichia kudriavzevii SJTU-BEL-1 is cultured for 72 hours, the dry weight of thalli reaches 41.25 + / -0.69 g / L, which is obviously superior to that of other strains. The strain has great application potential in single-cell protein production, and can still maintain high-yield protein capacity under the condition of high-density fermentation; the total protein content and the total amino acid content are higher than those of a common yeast fermentation product, and good industrial fermentation potential and application prospects are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Pichia kudrica, SJTU-BEL-1, that produces single-cell proteins and its applications. Background Technology

[0002] In recent years, with global population growth and changes in dietary structure, the demand for high-protein foods has continued to rise. Traditional animal protein sources (such as beef, pork, eggs, and fish) face significant challenges in terms of resource utilization efficiency, environmental impact, and animal welfare. Therefore, seeking alternative protein sources has become a hot research topic in the global agriculture, food, and bio-industry sectors.

[0003] Against this backdrop, single-cell protein (SCP), as an emerging protein source, has attracted widespread attention due to its high production efficiency, environmental friendliness, and ability to utilize low-quality raw materials such as agricultural by-products and industrial organic waste. SCP is a biomass source with high protein content and balanced nutritional components (such as amino acids, vitamins, and minerals) produced by microorganisms such as bacteria, yeast, and microalgae through microbial fermentation technology. It is widely used in animal feed, aquatic feed, and human nutritional supplements.

[0004] Among the many microorganisms that can be used for SCP production, yeast is one of the most commercially promising strains for SCP production due to its ease of cultivation, high fermentation efficiency, rich protein content, and high safety. For example, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) Saccharomyces cerevisiae ) and Kudriazvibichi yeast ( Pichia kudriavzevii Traditional yeast strains, such as [specific examples of yeast strains], have been widely studied and applied. However, when faced with complex or low-quality carbon source substrates, they often exhibit problems such as low fermentation efficiency, low yield, and unstable product quality, which restricts their promotion in large-scale production and industrial applications. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a single-cell protein-producing strain of Pichia kudrizis SJTU-BEL-1 and its applications. The Pichia kudrizis SJTU-BEL-1 provided by this invention maintains high protein production capacity even under high-density fermentation conditions; its total protein content and total amino acid content are higher than those of ordinary yeast fermentation products, demonstrating good industrial fermentation potential and application prospects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of *Pichia kudrica* that produces single-cell protein (…). Pichia kudriavzeviiSJTU-BEL-1, accession number CGMCC No.39111.

[0007] This invention provides a microbial preparation comprising the above-described Pichia pastoris SJTU-BEL-1.

[0008] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: inoculating a seed culture containing Pichia kudrica SJTU-BEL-1 as described in the above technical solution into a culture medium, and fermenting to obtain the microbial preparation.

[0009] Preferably, the culture medium is based on YCB medium and further contains 80-100 g / L glucose, 10-30 g / L peptone and 4-6 g / L L-lysine; the pH of the culture medium is 4.5-5.0.

[0010] Preferably, the culture medium is based on YCB medium and further contains 100 g / L glucose, 20 g / L peptone and 5 g / L L-lysine; the pH of the culture medium is 4.8.

[0011] Preferably, the fermentation conditions include: a temperature of 25~35℃, a rotation speed of 180~220 rpm, and a culture time of 70~80 h.

[0012] Preferably, the fermentation conditions include: a temperature of 30°C, a rotation speed of 200 rpm, and a culture time of 72 h.

[0013] Preferably, the volume ratio of the seed culture to the culture medium is 3:50; the viable count of Pichia pastoris SJTU-BEL-1 in the seed culture is 1×10⁻⁶. 5 CFU / mL.

[0014] Preferably, the volume ratio of the culture medium to the fermentation vessel is 1:10.

[0015] This invention provides the application of the above-described Pichia pastoris SJTU-BEL-1 or the above-described microbial preparation in the preparation of single-cell protein, animal feed, aquatic feed or human nutritional supplement products.

[0016] Beneficial effects: This invention provides a single-cell protein-producing Pichia pastoris strain, SJTU-BEL-1, with accession number CGMCC No. 39111. The SJTU-BEL-1 strain was obtained from overripe strawberry residue from a supermarket through initial screening, secondary screening, and identification. After 72 hours of cultivation, its cell dry weight reached 41.25 ± 0.69 g / L, significantly superior to other strains. It has great application potential in single-cell protein production, maintaining high protein production capacity even under high-density fermentation conditions; its total protein content and total amino acid content are higher than those of ordinary yeast fermentation products, demonstrating good industrial fermentation potential and application prospects.

[0017] Biological Preservation Instructions SJTU-BEL-1, classified as *Pichia kudrica*. Pichia kudriavzevii It was deposited on December 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39111. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The results of screening for strain SJTU-BEL-1 are shown below; where a represents yeast colonies isolated and cultured from environmental samples; b represents the preliminary purification results of selected yeast colonies on YCB-lysine agar; c represents colonies purified in the second purification; and d represents the completely purified yeast strain SJTU-BEL-1. Figure 2 The images show the microscopic morphology of strain SJTU-BEL-1 under stained and unstained conditions; where a represents the results under a bright-field microscope; b represents the observation results of budding yeast cells; c represents a single round to oval yeast cell dispersed in a suspension; d represents the results of methylene blue staining at low magnification; and e represents the results of methylene blue staining at high magnification. Figure 3 The images are methylene blue stained micrographs of strain SJTU-BEL-1; where a represents an optical micrograph at low magnification with a scale bar of 50 μm, and b represents an optical micrograph at high magnification with a scale bar of 100 μm. Figure 4 Phylogenetic tree of strain SJTU-BEL-1 based on ITS rDNA sequence. Detailed Implementation

[0020] This invention provides a strain of Pichia kudrica, SJTU-BEL-1, which produces single-cell protein, with accession number CGMCC No. 39111.

[0021] This invention, through systematic strain isolation, screening, performance evaluation, and molecular identification, ultimately screened out the superior strain *Pichia pastoris* SJTU-BEL-1, suitable for industrial production of high-value-added single-cell proteins. Compared to traditional yeast screening methods based on purified carbon sources, this invention uses decaying fruit residue as a screening substrate, which is widely available, low-cost, and conforms to the concept of a "circular economy." Utilizing waste rich in sugars and proteins as screening material avoids dependence on pure culture media, facilitating the high-value utilization of biomass resources. This invention employs YCB selective medium supplemented with L-lysine, effectively inhibiting the growth of non-yeast microorganisms and some non-target fungi, thereby improving the screening purity and efficiency of the target yeast. Kudria zwibichi yeast SJTU-BEL-1 exhibits good adaptability to low pH, high salinity, and complex organic substrates (such as effluent from anaerobic digesters), and is particularly suitable for treating organic matter in wastewater with high pollutant concentrations and converting it into biomass with high protein content.

[0022] The Kudria zweipie yeast SJTU-BEL-1 exhibits strong stress resistance and can grow stably in culture environments with inhibitory byproducts, making it suitable for applications such as industrial wastewater resource utilization, biofuel and feed production.

[0023] The Kudria zwei Pichia pastoris SJTU-BEL-1 not only possesses the characteristics of high yield, stability, and reproducibility, but also demonstrates application potential in the synthesis of various bioactive substances such as organic acids, ethanol, and enzymes.

[0024] The *Pichia pastoris* SJTU-BEL-1 strain provided by this invention achieved a cell dry weight of 41.25 ± 0.69 g / L after 72 h of cultivation, significantly superior to other strains. It shows great potential for application in single-cell protein production, maintaining high protein production even under high-density fermentation conditions; its total protein content and total amino acid content are higher than those of ordinary yeast fermentation products, demonstrating excellent industrial fermentation potential and application prospects.

[0025] This invention provides a microbial preparation comprising the above-described Pichia pastoris SJTU-BEL-1.

[0026] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: inoculating a seed culture containing Pichia kudrica SJTU-BEL-1 as described in the above technical solution into a culture medium, and fermenting to obtain the microbial preparation.

[0027] In one embodiment, the culture medium is based on YCB medium and also contains 80-100 g / L glucose, 10-30 g / L peptone and 4-6 g / L L-lysine; the pH of the culture medium is 4.5-5.0.

[0028] In one embodiment, the culture medium is based on YCB medium and also contains 100 g / L glucose, 20 g / L peptone and 5 g / L L-lysine; the pH of the culture medium is 4.8.

[0029] As one implementation method, the fermentation culture conditions include: a temperature of 25~35℃, a rotation speed of 180~220rpm, and a culture time of 70~80h.

[0030] As one implementation method, the fermentation conditions include: a temperature of 30°C, a rotation speed of 200 rpm, and a culture time of 72 h.

[0031] In one embodiment, the volume ratio of the seed culture to the culture medium is 3:50; the viable count of Pichia pastoris SJTU-BEL-1 in the seed culture is 1×10⁻⁶. 5 CFU / mL.

[0032] In one embodiment, the volume ratio of the culture medium to the fermentation vessel is 1:10.

[0033] This invention provides the application of the above-described Pichia pastoris SJTU-BEL-1 or the above-described microbial preparation in the preparation of single-cell protein, animal feed, aquatic feed or human nutritional supplement products.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a single-cell protein-producing Pichia pastoris strain SJTU-BEL-1 and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Preparation Example The selective culture medium YCB-Lysine has the following formula: glucose / maltose / corn syrup 10-30 g / L, yeast extract 10 g / L, L-lysine 5 g / L and agar 15 g / L; pH 4.8.

[0036] The YCB liquid medium used for rescreening is based on YCB medium and also contains 8% (w / v) glucose, 2% (w / v) peptone and 1% (w / v) L-lysine, with a pH of 4.8.

[0037] Example 1 1. Preliminary screening of bacterial strains Mix 500 g of overripe strawberry residue from a supermarket with 500 mL of sterile water, let stand for 3 h, and then filter. Take 1 mL of the filtrate and inoculate it onto a YCB-Lysine solid plate, and incubate at 30℃ for 48 h. Observe and detect the colony morphology (round, milky white), growth rate and diameter, and screen colonies with a diameter >5 mm, smooth edges and no hyphae.

[0038] Three strains (Ali-1, Ali-2, and Ali-3) were obtained through screening. Among them, strain Ali-2 exhibited the fastest growth rate and the largest colony diameter (5.8 ± 0.2 mm), indicating that it has superior metabolic activity and substrate utilization capacity. Figure 1 ).

[0039] 2. Secondary screening of strains The three strains obtained from the initial screening were inoculated into YCB liquid medium for secondary screening, with an inoculation volume of 10 μL. 5 CFU / mL, cultured in shake flasks at 30℃ (shake speed 200 rpm) for 72 h, with OD measured every 12 h. 600 Values ​​and cell dry weight (DCW). Results are shown in Table 1.

[0040] Table 1. Culture results of yeast isolates

[0041] The results showed that strain Ali-2 exhibited the highest biomass accumulation at all time points, reaching a dry weight of 41.25 ± 0.69 g / L after 72 h of culture, significantly superior to other strains. This result fully demonstrates the application potential of strain Ali-2 in single-cell protein production.

[0042] 3. Morphological identification Morphological and microscopic observations revealed that Ali-2 formed smooth, round, milky-white raised colonies on YCB-Lysine agar plates. Figure 2 Microscopic observation after methylene blue staining () Figure 3 The cells were oval to ellipsoidal in shape, ranging in size from 3 to 6 µm, and exhibited polygonal budding reproduction. No pseudohyphae or filamentous structures were observed, which is consistent with the typical morphological characteristics of Pichia yeast.

[0043] 4. Molecular biological identification The rDNA ITS region of strain Ali-2 was amplified and sequenced using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.1) and ITS4 (5'-TCCAGGGTGACCAAAAAATCA-3', SEQ ID NO.2), yielding a clear 580 bp band and a high-quality consensus sequence of 578 bp. Alignment analysis with the NCBI GenBank database using BLASTn showed that this sequence shared 99.3% similarity with multiple strains of Pichia kudriavzevii. Figure 4 This allowed for the confirmation of its species classification at the molecular level. Based on this, the isolate was formally identified and named... Pichia kudriavzevii SJTU-BEL-1, GenBank login number PV812543. Pichia kudriavzevii SJTU-BEL-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39111.

[0044] Example 2: Optimization of Culture Conditions Will Pichia kudriavzevii SJTU-BEL-1 cells were inoculated into YCB medium as the base medium and cultured in mediums containing different concentrations of components (Table 2). The cells were cultured in shake flasks at 30°C (shaking speed 200 rpm) for 72 h. The dry weight of the cells was measured, with three replicates for each group. The results are shown in Table 2.

[0045] Table 2. Culture media with different carbon-nitrogen ratios

[0046] The results showed that the strain reached its highest cell dry weight (DCW = 44.32 g / L) at a carbon-to-nitrogen ratio of 10:1 (group D), which was the optimal culture condition.

[0047] Example 3: High-density fermentation test and application evaluation Validation strains Pichia kudriavzevii The protein production capacity and stability of SJTU-BEL-1 under high-density culture conditions were initially tested in simulated industrial fermentation.

[0048] 1. Cultivation conditions Use a 500 mL Erlenmeyer flask, and fill it with 50 mL of liquid. Culture medium formulation: Based on YCB medium, it also contains 10% (w / v) glucose, 2.0% (w / v) peptone and 0.5% (w / v) L-lysine, with a pH of 4.8 and a volume of 10% (v / v). Culture parameters: 30℃, 200 rpm, 72 h; Inoculum size: 3 mL (initial viable cell concentration 1×10⁻⁶). 5 (CFU / mL) 2. Measurement Indicators Measurement at 72 h: OD 600 Value, dry weight of cells (DCW); total protein content (Lowry method) (Table 3); essential amino acid content (HPLC method), the results are shown in Table 4.

[0049] Table 3. Results of total protein content changes over time.

[0050] The results showed that the Saccharomyces cerevisiae strain Pichia kudriavzevii SJTU-BEL-1 exhibited highly efficient fermentation capabilities in terms of biomass growth and protein production. At 24 h, the strain showed active growth with high single-cell protein (SCP) content, indicating that it was in the growth-related protein production phase. At 48 h, biomass increased significantly, but the SCP percentage decreased, due to the dilution effect caused by rapid cell proliferation and the accumulation of non-protein cellular components.

[0051] At 72 h, biomass production tended to stabilize, while protein concentration increased significantly, leading to a recovery in SCP content. This indicates that the strain gradually transitioned from growth-related metabolism to protein accumulation metabolism, thus 72 h is the optimal culture period for SCP production. Extending the culture to 96 h resulted in reduced biomass and protein yield, demonstrating that prolonged fermentation is not beneficial. Overall, the results confirm that… Pichia kudriavzevii SJTU-BEL-1 can be used for the industrial production of single-cell proteins, with maximum protein yield achieved during a growth cycle of approximately 72 hours.

[0052] Table 4 Results of High-Density Fermentation Tests and Application Evaluation

[0053] The results showed that Pichia kudriavzevii The SJTU-BEL-1 strain can still maintain high protein production capacity under high-density fermentation conditions; the total protein content and total amino acid content are higher than those of ordinary yeast fermentation products, indicating that this strain has good industrial fermentation potential and application prospects.

[0054] amino acid components The yeast strains obtained by fermentation under optimal growth conditions (72 h) were analyzed using an automated amino acid analyzer. Pichia kudriavzeviiThe amino acid composition of SJTU-BEL-1 biomass was determined after acid hydrolysis. Amino acid concentrations were measured based on stem cell weight, and the results are shown in Table 5.

[0055] The results show that Pichia kudriavzevii SJTU-BEL-1 has a complete and balanced amino acid composition, including both protein-derived and non-protein-derived amino acids. Glutamic acid (Glu), alanine (Ala), glycine (Gly), and leucine (Leu) are the dominant amino acids, with glutamic acid being the primary amino acid, accounting for approximately 20-35% of the total free amino acids.

[0056] In addition, the biomass of this strain contains high concentrations of essential amino acids, such as leucine, isoleucine, valine, threonine, phenylalanine, lysine, and histidine. The relatively high proportion of essential amino acids indicates that the protein produced by this strain has good nutritional value and can therefore be used as animal feed, aquaculture feed, and human nutritional supplement.

[0057] Interestingly, this strain also accumulated a variety of non-protein amino acids, including γ-aminobutyric acid (GABA), β-alanine, and α-aminobutyric acid. These amino acids indicate that the strain possesses a unique nitrogen metabolism and stress adaptation mechanism, which may explain its tolerance to acidic environments and its ability to remain stable in industrial fermentation settings.

[0058] In summary, Pichia kudriavzevii The unique amino acid structure of SJTU-BEL-1 indicates that this strain not only produces high concentrations of protein, but also boasts high protein quality due to its balanced amino acid composition. These characteristics make it a promising candidate for industrial production of single-cell proteins and for applications in the food, feed, and biotechnology fields.

[0059] Table 5 Saccharomyces cerevisiae strains Pichia kudriavzevii Qualitative amino acid composition of SJTU-BEL-1

[0060] Note: The amino acid composition indicates that this strain has a balanced amino acid ratio, with a high content of essential amino acids and an abundance of non-protein amino acids, which proves that this strain has good nutritional value and industrial application potential.

[0061] The significance of major amino acids and their nutritional importance: Compared to traditional protein sources widely used in animal and human nutrition, *Saccharomyces cerevisiae* strains... Pichia kudriavzeviiThe dominant amino acids in SJTU-BEL-1, including glutamic acid, alanine, glycine, and leucine, have significant nutritional and functional implications. Glutamic acid is a major component of the amino acid composition, playing a central role in nitrogen metabolism and serving as a key precursor in the biosynthesis of other amino acids. In animal feed formulations, glutamic acid helps improve palatability and gut health; in human nutrition, it is closely related to protein digestibility and metabolic efficiency. The high content of branched-chain amino acids (BCAAs), particularly leucine, isoleucine, and valine, is especially important because these amino acids play a crucial regulatory role in protein synthesis and muscle metabolism in humans and animals. Leucine is particularly prominent, as it can activate the mTOR signaling pathway, thereby promoting muscle protein synthesis and supporting growth, maintenance, and recovery. Protein sources rich in BCAAs (such as fishmeal, soybean meal, and whey protein) are highly valued in the feed and food industries; therefore, the similar amino acid dominance in P. kudriavzevii SJTU-BEL-1 highlights its potential as a competitive alternative protein source.

[0062] Furthermore, the presence of alanine and glycine further enhances the functional quality of the protein. These two amino acids are involved in energy metabolism, collagen synthesis, and the maintenance of intestinal integrity, all of which are key factors affecting animal performance and human health. Their abundant content improves the overall biological value of the protein and supports the efficient utilization of amino acids.

[0063] Notably, the detection of non-protein amino acids (such as γ-aminobutyric acid, GABA) further distinguishes this strain from traditional microbial and plant protein sources. GABA plays a physiological role in regulating stress, metabolic regulation, and gut-brain axis signaling in animals and humans. Pichiakudriavzevii The naturally accumulated GABA in SJTU-BEL-1 suggests an inherent functional advantage, which may help improve stress resistance and promote health benefits when its biomass is used as feed or a nutrient.

[0064] In summary, the presence and dominant role of these nutritionally important amino acids indicate that... Pichia kudriavzevii The protein profile produced by SJTU-BEL-1 is not only abundant in quantity but also comparable in quality to, and in some respects superior to, conventional protein sources currently used in animal feed and human nutrition. This unique amino acid composition highlights the biological value and industrial relevance of this strain as a sustainable single-cell protein source.

[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pichia kudrica, which produces single-cell protein ( Pichia kudriavzevii SJTU-BEL-1, accession number CGMCC No.39111.

2. A microbial preparation, characterized in that, Includes the Kudria zwitterion yeast SJTU-BEL-1 as described in claim 1.

3. The method for preparing the microbial preparation according to claim 2, characterized in that, include: The seed culture containing the *Pichia kudrica* SJTU-BEL-1 as described in claim 1 was inoculated into a culture medium, and the microbial preparation was obtained by fermentation.

4. The preparation method according to claim 3, characterized in that, The culture medium is based on YCB medium and also contains 80-100 g / L glucose, 10-30 g / L peptone and 4-6 g / L L-lysine; the pH of the culture medium is 4.5-5.

0.

5. The preparation method according to claim 4, characterized in that, The culture medium is based on YCB medium and also contains 100 g / L glucose, 20 g / L peptone and 5 g / L L-lysine; the pH of the culture medium is 4.

8.

6. The preparation method according to claim 3, characterized in that, The fermentation conditions include: temperature 25~35℃, rotation speed 180~220 rpm, and culture time 70~80 h.

7. The preparation method according to claim 6, characterized in that, The fermentation conditions include: a temperature of 30°C, a rotation speed of 200 rpm, and a culture time of 72 h.

8. The preparation method according to claim 3, characterized in that, The volume ratio of the seed culture to the culture medium is 3:50; the viable count of Pichia pastoris SJTU-BEL-1 in the seed culture is 1×10⁻⁶. 5 CFU / mL.

9. The preparation method according to claim 3, characterized in that, The volume ratio of the culture medium to the fermentation vessel is 1:

10.

10. The use of the Kudria zweipichia SJTU-BEL-1 as described in claim 1, or the microbial preparation as described in claim 2, or the microbial preparation obtained by any one of the preparation methods described in claims 3 to 9, in the preparation of single-cell protein, animal feed, aquatic feed, or human nutritional supplement products.

Images