Optimization of Medium Composition and Culture Conditions for Cell Multiplication of a High Quality Milk Beer Fermentation Yeast (Kluyveromyces marxianus)

Abstract

Milk beer is a distinctive alcoholic milk drink created in China, and increasingly popular with consumers. Yeast is key factor to its fermentation production, and determines the flavor and quality. In this investigation, a high-quality yeast Kluyveromyces marxianus MJ1 suitable for milk beer fermentation was utilized in an optimization study of proliferation culture. Results showed that using the optimal medium composition: wort-based medium supplemented with molasses 2.94%, wheat bran 1.56%, CaCO3 0.21%; and applying the optimal culture conditions: 28 °C, rotation speed 220 rpm, inoculation concentration 1.5×10⁶ CFU·mL⁻¹, the viable count of MJ1 reached a maximum of 7.8×10⁸ CFU·mL⁻¹. Compared to YPD broth, the viable count of MJ1 increased to 4.11 times, while the raw material cost decreased to about 1/6 of the cost of YPD broth. The study achieved high-density proliferation culture of MJ1 through usage of low cost crop by-products; and laid the foundation for preparation of active freeze-dried powder for applications in the milk beer industry.

Introduction

Milk beer (MB) is a new type of beverage made from whole milk, skim milk, or whey that is achieved through fermentation by utilizing lactic acid bacteria and yeast in a two-step process. Besides having the characteristics of beer - low alcohol content, slight carbonation, and white foam - it also possesses the characteristics of a milk-beverage - sweet and sour, refreshing, and milky aroma. MB is nutritious (i.e. rich in protein/amino acids and vitamins) and is easy to absorb. MB also has the function of improving blood circulation, quenching thirst, and eliminating fatigue. Existing data shows that it becomes a consumer favorite following its introduction into a market, and that it has a very high market value and development space (Qi and Qu, 2007; Yang et al., 2007).

Yeast is a key microorganism for MB fermentation, which can contribute to the main flavor substances such as alcohols, acids, and esters. Its fermentation characteristics determine the quality and flavor of the MB (Wang et al., 2018). At present, there are not many enterprises producing MB in China, and the existing enterprises often use commercially available Saccharomyces cerevisiae, which leads to a similar taste, a single variety, and high sucrose content in MB (Li et al., 2008). Despite China's MB companies constantly evolving - as well as the innovation in beverage production technology - the strict, new regulations being applied to the food and beverage manufacturing industry are creating challenges with MB production. First, the fermentation process requires wort, hops, and other auxiliary materials, which complicates the process, prolongs the production cycle, and significantly increases the cost. Secondly, the product has a single flavor, high alcohol content, and heavy beer taste, making it difficult to meet the needs of consumers. These have greatly affected the competitiveness of the MB products on the beverage market.

Therefore, there is an urgent need for MB enterprises to find high-quality, milk-derived yeasts with rich metabolites, low alcohol yield, and that are suitable for direct fermentation of MB (Hu et al., 2017). Studies have shown that Kluyveromyces marxianus as a milk-derived yeast can efficiently ferment lactose, producing low alcohol and good aroma when applied in food fermentation (Li et al., 2013). Li et al. (Li et al., 2013) and Wang et al. (Wang et al., 2017) applied K. marxianus in MB fermentation, with the findings indicating that the strain performed superbly and thus can replace S. cerevisiae for the industrial production of MB.

In this study, a high-quality K. marxianus strain MJ1 isolated from Xinjiang wild kefir grains was utilized. In an earlier investigation it was tested in a MB fermentation and was compared to a commercial strain MJ (provided by cooperative companies). The test encompassed the quality index of the fermented product, including protein content, total solids, titratable acidity, ethanol content, total viable counts of coliforms, yeast and molds, as well as sensory evaluation of products, and analysis of volatile components. And the results showed that the product made by MJ1 meets the national and industry standards for MB, and the quality of its product was superior to MJ (Hu et al., 2017).

In order to further meet the requirements of large-scale production of MB, the preparation of yeast active dry powder has become an inevitable trend, which requires the strain to have a higher concentration of live organism. Therefore, the present investigation focused on the optimization of the medium composition and culture conditions to realize the high-concentration proliferation culture of K. marxianus strain MJ1. The study finding could provide reliable research basis for subsequent active dry powder production.

Materials and Methods

Materials    YPD broth was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Potato, soybean sprout, white granulated sugar, wort concentrate, molasses, corn flour, tapioca starch, wheat bran, rice bran, fish meal, soybean cake powder, soy flour, and peanut cake powder were bought from local supermarkets. Skim milk powder was obtained from Anchor, Fonterra Co-operative Group Ltd. (New Zealand). All chemical reagents were analysis grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Yeast extract was obtained from Oxoid Ltd. (England).

Activation of strain and preparation of seed solution K. marxianus    strian MJ1 was placed in a 250 mL Erlenmeyer flask containing 100 mL of sterilized YPD broth and cultured with a shaker (Fu-ma QYC 200, Shanghai Foma Experimental Equipment Co., Ltd., China) at 28 °C, 160 rpm for 24 h. The bacterial solution was then transferred to a new YPD broth for an additional 24 hours to complete activation. The activated strain was inoculated onto YPD broth at a ratio of 2% (v/v) and cultured at 28 °C and 160 rpm for logarithmic phase to obtain the seed solution of MJ1, and the viable count was noted.

Screening of basic medium    The seed solution of MJ1 was inoculated onto 4 different media: potato - dextrose broth (PDB (20% potato; 2% dextrose)), soybean sprout medium (20% soybean sprout; 2% dextrose), skim milk medium (12% skim milk powder; 7% white granulated sugar), and wort basic medium (16.67% wort concentrate (2 Brix)) at a concentration of 1×10⁶ CFU·mL⁻¹, and cultured at 28 °C and 160 rpm for 24 hours. The optical density value (OD600) was measured every 4 hours to plot the growth curve of MJ1. The un-inoculated medium was used as a blank control group, respectively, and each test was repeated thrice.

Screening and optimization of supplemental components of proliferation medium    Studies have shown that the composition of a medium directly affects the growth of yeast, especially the type and content of carbon, nitrogen and inorganic salts (Angulo-Montoya et al., 2019; Shariati et al., 2019). Therefore, on the basis of the optimal basal medium obtained, it is necessary to study the optimal carbon source, nitrogen source and inorganic salt of strain MJ1. Furthermore, cost of proliferation is also one of the necessary factors to consider for industrial fermentation. That said, under the premise of meeting the growth and proliferation requirements, low-cost materials such as agricultural product processing by-products may be preferred choice (Aini et al., 2018).

In order to screen for the optimal carbon source, different concentrations of glucose, sucrose, molasses, white granulated sugar, potato, corn flour, and tapioca starch were evaluated. The concentration of glucose and sucrose were set to 5, 10, 15, 20, 25 g·L⁻¹, and the remaining carbon sources were 15, 30, 45, 60, 75 g·L⁻¹.

Regarding screening for optimal nitrogen source, different concentrations and different types of nitrogen source materials were evaluated - wheat bran, rice bran, fish meal, soybean cake powder, soy flour, peanut cake powder, yeast extract and fish meal peptone as organic nitrogen source; and ammonium chloride, ammonium sulfate and ammonium nitrate as inorganic nitrogen sources. Among them, the concentration of wheat bran and rice bran was set to 7.5, 15, 22.5, 30, 37.5 g·L⁻¹, whereas the remaining organic nitrogen sources was 1.5, 3, 4.5, 6, 7.5 g·L⁻¹, and the inorganic nitrogen source was 0.4, 0.8, 1.2, 1.6, 2.0 g·L⁻¹.

To screen for optimal inorganic salts, different concentrations of KH₂PO₄, K₂HPO₄, MgSO₄, CaCO₃ and KCl were evaluated. The concentrations were set to 0.4, 0.8, 1.2, 1.6, 2.0 g·L⁻¹ for KH₂PO₄, K₂HPO₄, MgSO₄, and 1, 2, 3, 4, 5 g·L⁻¹ for CaCO₃ and KCl.

The seed solution of MJ1 was centrifuged at 4 °C, 8 000 rpm for 5 min, and the cells were collected, washed with sterile water, and re-suspended to obtain an inoculum suspension. Then, it was inoculated onto the aforementioned media at a concentration of 1×10⁶ CFU·mL⁻¹, and the viable cell counts were measured after culturing at 28 °C and 160 rpm for 18 hours. A blank control test was also set, and each test was repeated three times.

Based on the screening results, the optimal carbon and nitrogen source as well as inorganic salt were selected as the investigation factors. Using central combination design (CCD) of response surface methodology (RSM) (Arumugam and Kumar, 2017), a total of 20 experiments were conducted with three factors and three levels.

Optimization of culture conditions    The growth of yeast is also affected by the culture conditions, which mainly include liquid volume, initial pH, culture temperature, rotation speed and inoculum concentration (Hu, 2017).

First, the optimization experiment of liquid volume was carried out. Different volumes (30, 40, 50, 60, 70 and 80 mL in six 250 mL Erlenmeyer flasks) of proliferation medium of natural pH (pH 5.36) was measured, and the seed solution of MJ1 (1×10⁶ CFU·mL⁻¹) was inoculated respectively. The optimum liquid volume was obtained by measuring the viable count after incubation at 28 °C and 160 rpm for 18 h. Then, the optimization test of initial pH, culture temperature, rotation speed, and inoculum concentration were sequentially performed. Likewise, the test was carried out at six levels of initial pH (pH 4.5, 5, 5.36, 5.5, 6, and 6.5, adjusted with 1 M HCl or 1 M NaOH); culture temperature (24, 26, 28, 30, 32 and 34 °C), rotation speed (140, 160, 180, 200, 220 and 240 rpm), and inoculum concentration (0.1×10⁶, 0.5×10⁶, 1×10⁶, 1.5×10⁶, 2×10⁶ and 2.5×10⁶ CFU·mL⁻¹).

Based on the test data, the factors that had major influence on the growth of MJ1 were selected as the influencing factors for orthogonal test - L₉ (3⁴), and the experiment was carried out to obtain the optimum culture conditions.

Determination of the viable count    The viable count of the yeast was achieved by the plate-count method (Wang et al., 2016), and the result was expressed as the CFU·mL⁻¹. Each test was done three times at the same time, and the results were averaged.

Calculation of raw material cost of the proliferation medium    For cost comparison of optimized proliferation medium and conventional YPD broth, the raw material cost per liter of medium (excluding water) was calculated based on average commercial price.

Statistical analysis    The design and data analysis of RSM experiment were performed in the Design Expert Software (Version 8, State-Ease, USA). The plotting of figures was performed in the Origin Software (Version 8.0; Microcal Software Inc., Northampton, MA, USA), and the error bars of figures showed standard deviation (SD). The analysis of variance was performed using the SPSS Software (Version 16.0, SPSS Inc., Chicago, USA). The statistics significance was evaluated using Duncan's multiple's range test and P < 0.05 was taken as significant.

Results and Discussion

Counting of seed solution of strain MJ1    By determining, the average viable count of the MJ1 seed solution obtained was 1.9×10⁸ CFU·mL⁻¹.

Selection of basic medium    As shown in the growth curve (Fig. 1), OD600 reached a maximum of 0.899 in wort medium, followed by PDB of 0.745, and only reached 0.548 and 0.410 in skim milk medium and soybean sprout medium, respectively. The optical density (OD600) can indirectly monitor the growth of cells, and it is directly proportional to the concentration of microorganisms (Lobete et al., 2015). Therefore, it can be concluded that the wort medium is most beneficial to the growth of strain MJ1. Wort is processed from barley as the main raw material. It is cheap, easy to obtain, and rich in oligosaccharides, maltose, peptides, and amino acids, which can provide nutritional conditions for the growth of microorganisms (Chong et al., 2007). Finally, wort medium was considered the best basic medium (compared to the others).

Fig. 1.

Growth curve of MJ1 in four different basic medium

In these four media, MJ1 reached the logarithmic and stable phases of the growth curve almost simultaneously, where the logarithmic growth phase began around the 4^th hour and turned into the stable phase around the 16^th hour. The harvest time of the cells is preferably selected in the late logarithmic phase (the early stable phase) (Corcoran et al., 2010). Therefore, 18 hours of culture was taken as the optimal time for collection of strain MJ1 cells.

Selection of optimal carbon source for proliferation medium    The carbon source is the main source of nutrients and energy required by yeast. Sufficient carbon source must be provided during fermentation to ensure growth and proliferation of yeast. However, excessive concentrations of carbon sources can also inhibit yeast growth (Pereira et al., 2010). Different yeast strains have different optimal carbon source preference due to their different physiological metabolic pathways. Therefore, screening for the optimal carbon source and concentration has a positive effect on increases in growth rate as well as cell concentration.

For the test of glucose and sucrose, compared with the blank control, the viable count of MJ1 showed a decreasing trend as the glucose concentration increased, which may be affected by the Crabtree effect (JoRgensen, 2009). Unlike glucose, the viable count increased initially and then decreased with increasing sucrose concentration, and reached the highest value of 2.27×10⁸ CFU·mL⁻¹ at the concentration of 15 g·L⁻¹ (P < 0.05; Fig. 2A). Deducing from this is that, sucrose provided a more suitable osmotic pressure environment than glucose at a certain concentration, and thus could be more favorable for yeast growth. When molasses, white granulated sugar, potato and corn flour were used as carbon sources, the viable count increased initially and then decreased with increasing concentration. However, the addition of tapioca starch did not have positive effect on the proliferation of MJ1 within the range of concentrations tested (Fig. 2B). And the proliferation effect of molasses was the most significant, and the viable count reached a maximum of 3.42×10⁸ CFU·mL⁻¹ at 30 g·L⁻¹ (P < 0.05). At the respective optimum concentrations, molasses obtained a higher viable count than sucrose. As a by-product of the sugar industry, molasses contains a significant amount of sucrose and a small amount of other saccharides such as raffinose, glucose, lactose, etc. It provides a good carbon source for yeast cell growth and is considered an ideal low-cost material (Santos et al., 2010; Leelavatcharamas et al., 2018). Therefore, molasses was labelled as the optimal carbon source for the proliferation medium of strain MJ1.

Fig. 2.

Effect of carborn sources and concentration on the growth of MJ1

Different lowercase letters indicate significant difference (P < 0.05).

Selection of optimal nitrogen source for proliferation medium    Nitrogen is one of the essential components of cells and plays a vital role in the growth and development of yeast. Nitrogen sources are important sources of nitrogen-containing metabolites, mainly including organic nitrogen sources and inorganic nitrogen sources (Zhang et al., 2018). The organic nitrogen sources commonly used in the fermentation industry include wheat bran, rice bran, fish meal, soybean cake powder, peanut cake powder, peptone, yeast extract, etc., while the inorganic nitrogen source includes ammonium salts, nitrates, ammonia water, etc (Kang, 2015).

As a by-product of crop processing, wheat bran and rice bran contain ample amount of crude protein, fat, crude fiber and multivitamins, which can be used as the main nitrogen source in the fermentation process (Hammami et al., 2018). In the test of these two nitrogen sources, by comparison, the viable count of MJ1 reached the highest value of 2.83×10⁸ CFU·mL⁻¹ when the wheat bran concentration was 15 g·L⁻¹ (P < 0.05; Fig. 3A). In Fig. 3B, the soybean cake powder significantly increased the viable count of MJ1 within the tested concentration range, and the peanut cake powder had little effect, while the soy flour and fish meal caused the decrease in the viable count. The results of the comparison showed that the viable count reached a maximum of 2.75×10⁸ CFU·mL⁻¹ with adding 4.5 g·L⁻¹ soybean cake powder (P < 0.05). When the yeast extract and the fish meal peptone were used as the nitrogen source, with the concentration increasing, the viable count increases initially and then decreases, and reached the highest value of 2.37×10⁸ CFU·mL⁻¹ at 4.5 g·L⁻¹ yeast extract concentration (P < 0.05; Fig. 3C). At the respective optimal concentrations, compared with soybean cake powder and yeast extract, wheat bran had the best effect on increasing the viable count of MJ1. For the three inorganic nitrogen sources, their gradual addition resulted in a decrease in the viable count of MJ1 – suggesting a non-conducive condition for the proliferation of the strain (Fig. 3D). Thus, through the test of organic nitrogen source and inorganic nitrogen source, wheat bran was selected as the optimal nitrogen source for the proliferation medium of strain MJ1.

Fig. 3.

Effect of organic (A–C) and inorganic (D) nitrogen sources and concentration on the growth of MJ1 Different lowercase letters indicate significant difference (P < 0.05).

Selection of optimal inorganic salt for proliferation medium    During the growth and development of yeast, some inorganic salt ions are needed to regulate their physiological metabolic activities. For example, K⁺ and Ca²⁺ can regulate cell osmotic pressure and permeability, and Mg²⁺ can regulate intracellular enzyme activity (Xue et al., 2008).

In the test of KH₂PO₄, K₂HPO₄, MgSO₄, CaCO₃ and KCl as inorganic salts, the addition of the other four inorganic salts (except CaCO₃) resulted in a decrease in the viable count of the strain MJ1 as the concentration increased. The reason for this phenomenon may be that the strain has a low demand for inorganic salts, and the rich minerals in the wort are reasonably sufficient; and thus excess may not be suitable for the growth of the strain. On the contrary, the viable count was significantly increased after the addition of CaCO₃, and reached a maximum of 2.66×10⁸ CFU·mL⁻¹ at 2 g·L⁻¹ (P < 0.05; Fig. 4). This may be due to the fact that MJ1 produced some acidic substances that are not conducive to growth during fermentation, while CaCO₃ can play a role of neutralization regulation. Therefore, CaCO₃ was selected as the optimal inorganic salt for the proliferation medium of strain MJ1.

Fig. 4.

Effect of inorganic salts and concentration on the growth of MJ1

Different lowercase letters indicate significant difference (P < 0.05).

RSM modeling of optimum proliferation medium components    RSM is a statistical method used to find the optimal response factors and has been widely used to optimize the composition of culture medium (Coninck et al., 2000; Haddar et al., 2010; Yoo et al., 2018). Table 1 shows the experimental findings using the viable count as response variables. By applying the statistical technique - multiple regression analysis on the experimental data, the following second-order polynomial equation giving the viable count (Y) as a function of molasses (A), wheat bran (B) and CaCO₃ (C) concentrations was obtained:   

Table 1. Central combination design (CCD) and results of the viable count

Run	Independent variables (uncoded value)			Dependent variables
	A (%)	B (%)	C (%)	The viable count (×108 CFU·mL−1)
1	3	1.5	0.2	7
2	1.5	0.75	0.3	4.6
3	0.48	1.5	0.2	4.6
4	3	1.5	0.03	5.2
5	4.5	2.25	0.3	5
6	3	1.5	0.2	6.7
7	1.5	2.25	0.1	4.9
8	1.5	0.75	0.1	4.8
9	5.52	1.5	0.2	4.3
10	3	1.5	0.37	5.4
11	3	1.5	0.2	7
12	4.5	2.25	0.1	4.6
13	3	1.5	0.2	6.9
14	4.5	0.75	0.1	4.5
15	4.5	0.75	0.3	4.8
16	3	0.24	0.2	4.8
17	3	2.76	0.2	5.1
18	1.5	2.25	0.3	5.1
19	3	1.5	0.2	6.8
20	3	1.5	0.2	6.8

The ANOVA analysis (Table 2) revealed that the model is extremely significant (P < 0.0001); the parameters of A, B, C, AC, BC, A², B², and C² had significant effects on the proliferation of strain MJ1 (P < 0.05); the model mismatch term is not significant (P > 0.05); the correction coefficient R² and correlation coefficient R² are 0.9954 and 0.9976, respectively. This model can thus be used to analyze changes in response values.

Table 2. Variance analysis of the response surface quadratic model for the viable count

Source	Sum of square	df	Mean square	F-value	P-valuea
Model	19.36	9	2.15	461.07	< 0.0001
A-Molasses	0.074	1	0.074	15.84	0.0026
B-Wheat bran	0.14	1	0.14	30.96	0.0002
C-CaCO3	0.079	1	0.079	16.86	0.0021
AB	0.011	1	0.011	2.41	0.1515
AC	0.061	1	0.061	13.13	0.0047
BC	0.031	1	0.031	6.70	0.0270
A2	10.91	1	10.91	2337.95	< 0.0001
B2	6.93	1	6.93	1484.39	< 0.0001
C2	4.67	1	4.67	1001.76	< 0.0001
Residual	0.047	10	4.67E-03
Lack of Fit	0.018	5	3.67E-03	0.65	0.6779
Pure Error	0.028	5	5.67E-03
Cor Total	19.41	19
	R2=0.9976		R2(adj)=0.9954

^a  The P-values less than 0.05 are signifcant, and the P-values less than 0.01 are extremely significant.

The model predicted that when the concentration of molasses, wheat bran and CaCO₃ were 2.94%, 1.56% and 0.21%, respectively, the viable count would reach the highest value of 6.9×10⁸ CFU·mL⁻¹. Three independent experiments were conducted at this concentration, and the actual viable count was 6.8×10⁸ CFU·mL⁻¹, which was very close. It can be inferred that the parameters of the proliferation medium components obtained by the RSM modeling are accurate and reliable. Moreover, the viable count was about 3.58 times that of YPD broth (1.9×10⁸ CFU·mL⁻¹), which indicated that the optimized medium had a remarkable proliferative effect. The above results laid the experimental basis for optimizing the culture conditions to further increase the viable count of the strain MJ1.

Effects of different culture conditions on the growth of strain MJ1    The amount of liquid in the microbial shake flask culture process can affect the oxygen transfer, and many studies have tested the optimal liquid volume (Liu and Yang, 2006; Xu et al., 2010; Qi, 2014; Jiang et al., 2018). If the liquid volume is too low, the water will easily evaporate and the test error will be large. Conversely, if the volume of liquid is too high, the amount of dissolved oxygen will be insufficient, which is not conducive to the proliferation and culture of microorganisms. Most yeast grow optimally in the pH range 4.5 ∼ 6.5, and thus too high or too low will affect their growth and proliferation (Yin et al., 2010; Guo et al., 2019). Temperature has a significant effect on the growth, proliferation, and metabolic activity of microorganisms. A certain increase in temperature can increase the activity of intracellular enzymes and membrane fluidity, as well as the sensitivity of cells to alcohol toxicity (Reddy and Reddy, 2011). The rotation speed directly determines the level of oxygen supply in the medium. If the rotation speed is too low, the dissolved oxygen content of the medium will be lowered, which is not conducive to the absorption and metabolism of nutrients by the strain. On the contrary, excessively high rotation speed can cause high shear forces on cells and inhibit cell growth (Garcia-Ochoa et al., 2010). A suitable inoculum concentration is vital for the proliferation of the strain. Insufficient inoculum will prolong the time to reach the stable phase, and excessive inoculum will result in insufficient nutrient in the medium to affect the metabolism and proliferation of the cells (Singh et al., 2007).

The effects of different culture conditions (liquid volume, initial pH, temperature, rotation speed, and inoculum concentration) on the proliferation of MJ1 were shown in Fig. 5. The viable count showed a trend of increasing initially and then decreasing with the change of certain condition, which was consistent with the above description. And at temperature 28 °C, rotation speed 220 rpm, and inoculation concentration 1.5×10⁶ CFU·mL⁻¹, the viable count of MJ1 reached the highest value (P < 0.05), respectively. For the liquid volume, although the viable count achieved the maximum at 50 mL, the difference was not significant in the range of 40 mL to 60 mL (P > 0.05). Similarly, the viable count reached the highest value at pH 5.36, but the difference was not significant within pH 5.0 ∼ 5.36 (P > 0.05). Therefore, the liquid volume and pH were not suitable as the influencing factors for the orthogonal.

Fig. 5.

Effect of liquid volume (A), pH (B), temperature (C), rotation speed (D) and inoculum concentration (E) on the growth of MJ1 Different lowercase letters indicate significant difference (P < 0.05).

Optimization of culture conditions by orthogonal test    The culture temperature, rotation speed and inoculum concentration that had significant influence on the MJ1 growth were selected as the influencing factors for the orthogonal [L₉ (3⁴)] test. From Table 3 and Table 4, the effects of factors A (temperature), B (rotation speed) and C (inoculation concentration) on the viable count of MJ1 were R_A > R_B > R_C. Except for the inoculation concentration, the other factors had significant effects on the viable count (P < 0.05). The optimal growth condition for strain MJ1 was A₂B₂C₂ (temperature 28 °C, rotation speed 220 rpm, and inoculation concentration 1.5×10⁶ CFU·mL⁻¹). Three independent experiments were done under the optimal conditions, and the average viable count was 7.8×10⁸ CFU·mL⁻¹, which was higher than the optimal combination A₂B₂C₃ of 7.6×10⁸ CFU·mL⁻¹ in the orthogonal experiment. And the test had good repeatability, which indicated A₂B₂C₂ can be used as the optimal culture condition for strain MJ1. Finally, the viable count of strain MJ1 obtained after optimization of proliferation culture was as high as 7.8×10⁸ CFU·mL⁻¹, which was about 4.11 times that of YPD broth (1.9×10⁸ CFU·mL⁻¹), and the effect was remarkable.

Table 3. Results of orthogonal test for culture conditions

Run	Levels of coded (uncoded) value				The viable count (Y, ×107 CFU·mL−1)
	Temperature (A, °C)	Rotation speed (B, rpm)	Inoculum concentration (C, ×106 CFU·mL−1)	Blank (D)
1	1(26)	1(200)	1(1.0)	1	62
2	1(26)	2(220)	2(1.5)	2	71
3	1(26)	3(240)	3(2.0)	3	65
4	2(28)	1(200)	2(1.5)	3	72
5	2(28)	2(220)	3(2.0)	1	76
6	2(28)	3(240)	1(1.0)	2	74
7	3(30)	1(200)	3(2.0)	2	64
8	3(30)	2(220)	1(1.0)	3	71
9	3(30)	3(240)	2(1.5)	1	67
K1	198	198	207	205
K2	222	218	210	209
K3	202	206	205	208
k1	66	66	69	68.333
k2	74	72.667	70	69.667
k3	67.333	68.667	68.333	69.333
R	8.000	6.667	1.667	1.334
Factor order	A > B > C > D
Optimal level	2	2	2
Optimum	A2B2C2
combination

Table 4. ANOVA analysis of orthogonal test

Source	Sum of squares	df	Mean square	F-value	P-valuea
Temperature	110.222	2	55.111	38.154	0.026
Rotation speed	67.556	2	33.778	23.385	0.041
Inoculum concentration	4.222	2	2.111	1.462	0.406
Error	2.889	2	1.444

^a  The P-values less than 0.05 are signifcant, and the P-values less than 0.01 are extremely significant.

Comparison of the cost of the optimal proliferation medium and YPD broth    The optimal proliferation medium consisted of wort base medium (16.67% wort concentrate), molasses 2.94%, bran 1.56% and CaCO₃ 0.21%. Reference to the average commercial price, the unit price (per kilogram) of wort concentrate, molasses, wheat bran, and CaCO₃ are 23, 10, 4, and 8 Yuan, respectively, therefore it costs 4.21 Yuan per liter of proliferation medium. The unit (per kilogram) price of YPD broth is 548 yuan, and it costs 27.4 Yuan per liter of medium (50 g·L⁻¹), meaning the cost of the proliferation medium optimized was less than 1/6 of YPD broth. This study not only achieved high-density fermentation of strain MJ1, but also significantly reduced the cost of proliferation and improved the utility value of agricultural product processing by-products. Moreover, this laid the foundation for further mass cultivation of MJ1 live cells using fermenters.

Conclusion

K. marxianus strain MJ1 suitable for milk beer fermentation production was used in an optimization study of proliferation culture. Using the optimized proliferation medium (molasses 2.94%, wheat bran 1.56%, CaCO₃ 0.21%) and applying the optimal culture conditions (28 °C, rotation speed 220 rpm, inoculation concentration 1.5×10⁶ CFU·mL⁻¹), the viable count of strain MJ1 increased from 1.9×10⁸ CFU·mL⁻¹ (YPD broth) to 7.8×10⁸ CFU·mL⁻¹, which was increased to 4.11 times. In addition, this study utilized crop by-products as a raw material for proliferation, with a total cost of 4.21 Yuan per liter. And the price of YPD broth was 27.4 Yuan per liter, which represented nearly 5 times reduction in cost.

The results set a good foundation for further mass production of the strain MJ1 active cells, as well as the preparation of the active freeze-dried powder and the industrial application of the strain MJ1 in MB production.

Acknowledgements    This work was supported by Jiangsu University senior professional talent research start-up funds (12JDG069), Xinjiang Production and Construction Corps Industry and High-tech Science and Technology Research and Achievement Transfer Project (2015AB032) and Xinjiang Production and Construction Corps 12th Division Industrial Research Project (SR2016001, SR2017003).

References

•  Aini,  F.N.,  Laconi,  E.B., and  Suhartono,  M.T. (2018). The Potential of Agricultural By-products as a Carbon Source in the Growth of Paenibacillus sp.. Presented at the 8th Annual Basic Science International Conference: Coverage of Basic Sciences Toward the World's Sustainability Challanges, Melville, October 17, pp. 1-5.
•  Angulo-Montoya,  C.,  Ruiz Barrera,  O.,  Castillo-Castillo,  Y.,  Marrero-Rodriguez,  Y.,  Elias-Iglesias,  A.,  Estrada-Angulo,  A.,  Contreras-Perez,  G.,  Arzola-Alvarez,  C., and  Carlos-Valdez,  L. (2019). Growth of Candida norvegensis (strain Levazoot 15) with different energy, nitrogen, vitamin, and micromineral sources. Braz. J. Microbiol., 50, 533-537.
•  Arumugam,  T. and  Kumar,  S.P. (2017). Optimization of media components for production of antimicrobial compound by Brevibacillus brevis EGS9 isolated from mangrove ecosystem. J. Microbiol. Methods, 142, 83-89.
•  Chong,  K.,  Tian,  H.T.,  Sang,  Y.X.,  Li,  Y.J.,  Ma,  X.Y., and  Pang,  X.N. (2007). Optimization of Reproducible Wort Medium for Bifidobacteria. FOOD SCIENCE, 28, 178-182 (in Chinese).
•  Coninck,  J.D.,  Bouquelet,  S.,  Dumortier,  V.,  Dyume,  F., and  Verdier-Denantes,  I.. (2000). Industrial media and fermentation processes for improved growth and protease production byTetrahymena thermophilaBIII. J. Ind. Microbiol. Biotechnol., 24, 285-290.
•  Corcoran,  B.M.,  Ross,  R.P.,  Fitzgerald,  G.F., and  Stanton,  C. (2010). Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances. J. Appl. Microbiol., 96, 1024-1039.
•  Garcia-Ochoa,  F.,  Gomez,  E.,  Santos,  V.E., and  Merchuk,  J.C. (2010). Oxygen uptake rate in microbial processes: An overview. Biochem. Eng. J., 49, 289-307.
•  Guo,  Q.,  Zabed,  H.,  Zhang,  H.H.,  Wang,  X.,  Yuan,  J.H.,  Zhang,  G.Y.,  Yang,  M.M.,  Sun,  W.J., and  Qi,  X.H. (2019). Optimization of fermentation medium for a newly isolated yeast strain (Zygosaccharomyces rouxii JM-C46) and evaluation of factors affecting biosynthesis of D-arabitol. LWT--Food Sci. Technol., 99, 319-327.
•  Haddar,  A.,  Fakhfakh-Zouari,  N.,  Hmidet,  N.,  Frikha,  F.,  Nasri,  M., and  Kamoun,  A.S. (2010). Low-cost fermentation medium for alkaline protease production by Bacillus mojavensis A21 using hulled grain of wheat and sardinella peptone. J. Biosci. Bioeng., 110, 288-294.
•  Hammami,  A.,  Bayoudh,  A.,  Abdelhedi,  O., and  Nasri,  M. (2018). Low-cost culture medium for the production of proteases by Bacillus mojavensis SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products. Ann. Microbiol., 68, 473-484.
•  Hu,  M. (2017). Screening of High Quality Dairy Yeast for Milk Beer Producing and Studies on Active Dry Yeast Preparation. M. Sc. Thesis, Jiangsu University, Zhenjiang.
•  Hu,  M.,  Wang,  L.,  Gong,  X.F.,  Wang,  J.Y.,  Liu,  P.L.,  Zhan,  T., and  Li,  Y. (2017). Study on screening of high quality dairy yeast for milk beer producing and fermentation characteristics. Dairy Industry, 45, 27-32 (in Chinese).
•  Jiang,  A.T.,  Li,  X.L.,  Jiang,  S.J.,  Tuo,  Y.F.,  Qian,  F., and  Ma,  F.L. (2018). Optimizing on proliferation conditions of preferred Saccharomyces cerevisiae and construction of fermentation kinetics model. Journal of Food Science and Technology, 36, 45-52. (in Chinese).
•  JoRgensen,  H. (2009). Effect of Nutrients on Fermentation of Pretreated Wheat Straw at very High Dry Matter Content bySaccharomyces cerevisiae. Appl. Biochem. Biotechnol., 153, 44-57.
•  Kang,  Y.J. (2015). High-cell-density cultivation of a salt-tolerant yeast Zygosaccharomyces rouxii. M. Sc. Thesis, Hubei Technology University, Xianning.
•  Leelavatcharamas,  V.,  Sritongon,  K.,  Salakkam,  A., and  Kishida,  M. (2018). Optimization of lipid accumulation in thermotolerant yeast Candida sp Sbc 06 using molasses as carbon source. New Biotechnol., 44, S121-S121.
•  Li,  X.L.,  Zhang,  D.F.,  Lu,  D.L.,  Yang,  X.Q., and  Qi,  Q.G. (2008). Study on processing technology and quality control low sugar yoghurt-beer drink. Dairy Industry, 36, 34-36 (in Chinese).
•  Li,  X.L.,  Gu,  R.X.,  Yan,  H.,  Zhang,  L.L., and  Zhang,  J.L. (2013). Screening, identification and application of Kluyveromyces cerevisiae. Milk Processing, 15, 44-46 (in Chinese).
•  Liu,  Y. and  Yang,  Z.H. (2006). Uses for chrome leather waste - Chromium enriched brewers yeast. J. Soc. Leather Technol. Chem., 90, 14-18.
•  Lobete,  M.M.,  Fernandez,  E.N., and  Van Impe,  J.F.M. (2015). Recent trends in non-invasive in situ techniques to monitor bacterial colonies in solid (model) food. Frontiers in Microbiology, 6, 148.
•  Pereira,  F.B.,  Guimaraes,  P.M.R., and  Teixeira,  J.A. (2010). Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs. Bioresour. Technol., 101, 7856-7863.
•  Qi,  H.L. (2014). The Candida utilis fermentation proliferation and post treatment studies. M. Sc. Thesis, Shihezi University, Shihezi.
•  Qi,  Q.G. and  Qu,  H.Q. (2007). Research and Development of Milk Beer Beverage. New Products and Technology, 3, 24-25 (in Chinese).
•  Reddy,  L. and  Reddy,  O. (2011). Effect of fermentation conditions on yeast growth and volatile composition of wine produced from mango (Mangifera indica L.) fruit juice. Food Bioprod. Process, 89, 487-491.
•  Santos,  A.C.,  Bezerra,  M.S.,  Pereira,  H.S.,  Santos,  E.S., and  Macedo,  G.R. (2010). Production and Recovery of Rhamnolipids Using Sugar Cane Molasses as Carbon Source. J. Chem. Chem. Eng., 4, 27-33.
•  Shariati,  S.,  Zare,  D., and  Mirdamadi,  S. (2019). Screening of carbon and nitrogen sources using mixture analysis designs for carotenoid production by Blakeslea trispora. Food Sci. Biotechnol., 28, 469-479.
•  Singh,  R.S.,  Sooch,  B.S., and  Puri,  M. (2007). Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour. Technol., 98, 2518-2525.
•  Wang,  L.,  Gao,  E.Y.,  Hu,  M.,  Oladejo,  A.,  Gong,  X.F.,  Wang,  J.Y., and  Zhong,  H. (2018). Isolation, identification and screening of high-quality yeast strains for the production of milk beer. Int. J. Dairy Technol., 71, 944-953.
•  Wang,  L.,  Zhong,  H.,  Liu,  K.,  Guo,  A.,  Qi,  X., and  Cai,  M. (2016). The evaluation of kefir pure culture starter: Liquid-core capsule entrapping microorganisms isolated from kefir grains. Food Sci. Technol. Int., 22, 598.
•  Wang,  W.,  Zhang,  K.,  Zhang,  Y.N.,  Wu,  H.T.,  Tian,  G.,  Hua,  Y., and  Wu,  Y. (2017). Optimization and stability prediction on the fermentation condition of koumiss beer by Box-Behnken method. Food Science and Technology, 42, 99-105 (in Chinese).
•  Xu,  C.,  Chen,  L.J., and  Shi,  W.C. (2010). Research on the optimization of fermentation conditions for Kluyveromyces lactis and properties of intracellular lactase. China Food Additives, 128-133 (in Chinese).
•  Xue,  C.,  Zhao,  X.Q.,  Yuan,  W.J., and  Bai,  F.W. (2008). Improving ethanol tolerance of a self-flocculating yeast by optimization of medium composition. World J. Microbiol. Biotechnol., 24, 2257-2261.
•  Yang,  H.M.,  Liu,  W.H.,  Xu,  L.Q.,  Li,  Y.,  Xu,  X.J., and  Zhang,  W. (2007). Study on the milk beer beverage. Food and Machinery, 23, 111-113 (in Chinese).
•  Yin,  H.,  Fan,  G., and  Gu,  Z. (2010). Optimization of culture parameters of selenium-enriched yeast (Saccharomyces cerevisiae) by response surface methodology (RSM). LWT--Food Sci. Technol., 43, 0-669.
•  Yoo,  H.,  Rheem,  I.,  Rheem,  S., and  Oh,  S. (2018). Optimizing Medium Components for the Maximum Growth of Lactobacillus plantarum JNU 2116 Using Response Surface Methodology. Korean J. Food Sci. Anim. Resour., 38, 240-250.
•  Zhang,  W.P.,  Du,  G.C.,  Zhou,  J.W., and  Chen,  J. (2018). Regulation of Sensing, Transportation, and Catabolism of Nitrogen Sources in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev., 82, e00040-17.