Cultivation of Aspergillus oryzae and Saccharomyces cerevisiae in Whey for the Production of Single-celled Protein Intended for Feeding Cattle

The nutritional capacity of lactoserum allows the cultivation of microorganisms such as Aspergillus oryzae and Saccharomyces cerevisiae, which have probiotic properties for cattle. The objective of this work was to evaluate the use of whole lactoserum as a culture medium for the production of biomass of these microorganisms under different conditions and types of culture. The results obtained from the characterization of lactoserum indicate similarity between those reported by other authors for whey of different origins. An experimental design of three factors and two levels was carried out in a model system, where the temperature of pasteurization and/or sterilization was evaluated at 60 and 120° C respectively; initial pH of 4 and 7; as well as the use of yeast extract, 0 and 10 g/L. The best results were obtained at 60°C, pH=7 and in the absence of yeast extract for A. oryzae and in the presence of this for the cultivation of S. cerevisiae. These conditions were used for crops, obtaining maximum biomass values of 6.5 g/L for axenic crops of S. cerevisiae and A. oryzae, 4.80 g/L for mixed cultivation, 22.25 g/L and 15.0 g/L respectively; the latter highlighting the latter for allowing greater productivity.


Introduction
Whey, whey or cheese whey is the liquid resulting from the coagulation of milk during cheese making. In general, there are 2 types of whey according to the cheese making process: acid whey and sweet whey, the latter corresponding to the highest-producing whey in Mexico [1]. This byproduct of the milk industry is often used, in small quantities, for the feeding of different types of livestock and the rest is discarded in the effluents causing pollution to the environment [2,3]. According to what is reported by Badui [4], for every 10 liters of milk, 8 to 9 kg of whey is produced and according to the Ministry of Agriculture, Livestock and Rural Development, Fisheries and Food (SAGARPA) during the year 2018 11,607,493,000 liters of milk and 342,000 tons of cheese per year were produced nationwide; Likewise, a production of 448,782,000 liters of milk was reported in the state of Puebla. Following that trend, in that year approximately 5 475 tons of cheese were produced in the state, which translates into an approximate production of 4.4 million liters of whey per year. Currently, whey production is a problem due to its high pollutant capacity, a consequence of its buffering properties of pH, proteins, lactose and other nutrients that make it an excellent culture medium for a wide variety of bacteria, yeasts and fungi. In the world there are different technologies for the reuse of whey, from dehydrated whey, flavored drinks, fermented products to protein concentrates. The present investigation focused on the production of single-cell protein of Aspergillus oryzae and Saccharomyces cerevisiae by fermentation of whole serum. Due to the high nutritional value of the unicellular protein and the probiotic effects attributed to some microorganisms, the use of fungi and yeasts as animal feed has become a topic of interest for the animal feed industry. Based on the above,

Background
The dairy industry is in third place in terms of importance in the activities in the Mexican industrial branch. According to statistics provided by SAGARPA during 2016, 11,607,493,000 liters of milk were produced in the country, placing it in the ninth position worldwide. Regarding the current situation of the dairy sector in the state of Puebla, in the same year 448,782,000 liters were produced, placing the state in eighth place nationally [5]. Likewise, the production of different types of cheeses reached the figure of 342,000 national annual tons and 5,475 state tons [6]. Regarding livestock production, according to data provided by the National Institute of Statistics and Geography, in 2014, there were 28 415 337 head of cattle in the country of which 379,595 heads correspond to the state of Puebla and that place it in the 22nd place in the national ranking [7,8]. Based on these figures, it is estimated that the national annual production of whey is of approximately 273.6 million liters and 4.4 million liters in the state of Puebla.
The Agrifood and Fisheries Information Service (SIAP) reports that, in Puebla, at the end of 2016 in the Cholula region, 120,765,000 liters of milk were produced, representing about 27% of state production. The main producing municipalities were San Matías Tlalancaleca, Ocoyucan, San Gregorio Atzompa and San Martín Texmelucan with productions greater than 9 thousand liters per year and concentrated about 10% of state production [9]. This information allows to locate in this area a niche of opportunity for the transformation of the whey into valueadded products.

Carbohydrates
Lactose in bovine milk is found in concentrations between 4.5% and 5.2%. This is a disaccharide formed by a glycoside bond (1,4) between a galactose molecule and another of glucose. It is much less sweet than sucrose, and together with salts, it contributes to overall taste of milk. It is a reducing sugar prone to enzymatic hydrolysis by the enzyme ∝galactosidase produced by a wide variety of microorganisms (Lactococcus, Lactobacillus, Kluyveromyces, Candida, Aspergillus, Mucor, etc.) and in the small intestine of mammals [4,1].

Proteins
In general, there are two types of milk proteins: fat globule membrane proteins and skim milk proteins. Firsts are absorbed within a pellicle that surrounds the fatty blood cells and which are associated with some enzymatic functions, whose function is to release fat and, sometimes, this disrupt the properties of some milk by-products such as the solubility of powdered milk [10].
From all proteins contained in it, 80% corresponds to casein, and the remaining 20% corresponds to whey proteins. The latter are considered soluble even in acidic conditions, while casein coagulates at pH less than 4.6.

Lipids
Milk fat fraction is mainly form by triacylglycerides (96-98%), although there are other lipids such as diacylglycerides, mono acylglycerides, phospholipids, free fatty acids, cholesterol and traces of some hydrocarbons like esters of sterols. These compounds are found in emulsion form, and they are responsible for some properties of milk, such as taste, nutritional and caloric requirements. Furthermore, they are an excellent means of transport to vitamins and other fat-soluble compounds [4].

Vitamins and Mineral Salts
The main vitamins contained in milk are Rivoflavin (vitamin B2), vitamin A, vitamin D and thiamine (vitamin B1), although it also has the rest of the necessary vitamins in the human diet.
The fat-soluble vitamins are transported through chemical interactions with the lipids present, while the rest, travels through the water; therefore, water-soluble vitamins are found in whey obtained from cheese making ( Table 1).
As far as salts are concerned, the main mineral in milk is calcium, followed by phosphorus, chlorides, potassium, sodium, magnesium and others such as iron and copper. Sodium and potassium chlorides are completely dissolved in the aqueous phase of milk, while calcium, magnesium and citrate phosphates form colloidal complexes with casein [10].

Milk Derivatives
Currently, in Mexico and in much of the world, bovine milk is the most consumed and from it a large variety of dairy products are produced that result from different processes; whether by physical transformations that allow extending the shelf life of the milk itself or by chemical or biological induced transformations for the generation of milk derivatives. Physical transformations allow immediate consumption products such as powdered milk, sweetened condensed milk and evaporated milk [11]. Among the induced transformations are coagulation, whip and fermentation to obtain cheese, butter and yogurt, respectively.
Milk fermentation is a widespread practice throughout the world, it is a process in which a large variety of microorganisms producing ∝-galactosidase are involved. Some products obtained from fermentation are yogurt, jocoque (drink or dessert made from soured milk), kefir, Bulgarian and "Yakult" [1]. Generally, this process is accompanied by the addition of other ingredients or food additives, in order to improve its physicochemical properties.
As a consequence of the biochemical transformations that happened in lactic cultures, they contain supplemental nutritional requirements such as B vitamins complex, an increase in the concentration of proteins and partially hydrolyzed lactose that are easily digested. Therewith, a diagram with the various processes to which milk and byproducts are subjected, is shown in Figure 1

Physicochemical and Biological Characteristics
During cheese making, proteins and fats are concentrated in a process called coagulation, then they are separated and compressed with the aim of giving consistency to cheese. The residue of this process is whey, which is considered a waste from the cheese industry. The fraction of milk that is transformed into cheese is only 10% to 20%, therefore, whey constitutes up to 90% of the total volume and contains up to 55% of the total nutrients of whole milk. Among the main components of the whey are: lactose, proteins, fats and mineral salts. In Table 2 [11] it can be observed that the serum is divided into two types according to its acidity but that in general there are no considerable differences in its chemical composition. As noted, the lactose and protein content is in high amounts, these confer a high organic content and a C/N ratio of intermediate value that make it a biodegradable substrate, in the same sense, this relationship is a fundamental factor in the development of any organism and will determine whether these nutrients will be consumed for the production of biomass or for the formation of biomolecules and other byproducts of fermentation [12][13][14]. Source: Own elaboration.

Alternatives for Lactoserum Reuse
Due to lactoserum is a highly polluting waste product, multiple applications have been studied in other areas than the dairy industry. Figure 1 shows some products obtained from whey using different procedures and several of them are described below. Whole whey can be used directly in liquid form as food for different types of farm animals, water can also be removed by evaporation and obtained a concentrated whey or dry it thoroughly and marketed in powder form. When the protein fraction is desired, it is advisable to subject lactoserum to ultrafiltration to separate the proteins from the rest of the solution and to maintain their properties. It is a high-cost process, but at the same time it generates a product of great added value.

Unicellular Protein Production
The generation of bacterial cells, filamentous fungi and yeasts by fermentation in different types of culture medium and under controlled conditions is what, in microbiology, is Whey for the Production of Single-celled Protein Intended for Feeding Cattle known as biomass or single-celled protein. This name is derived from the fact that single-celled organisms have high protein proportions in relation to their size and the presence of other cellular components.
Furthermore, single-celled protein contains lipids, carbohydrates, vitamins and minerals; as well as essential amino acids, such as lysine or methionine, which are found in limited amounts in plant and animal tissues [15]. Singlecelled protein production is born in response to two needs: counteracting the scarcity of protein sources for food and the use of wastes rich in fermentable sugars.
The presence of different types of carbohydrates in a large number of industrial by-products represents a great opportunity for the development of a wide range of microorganisms. Which may grow in such waste whether they require previous treatment (hydrolysis) or not. In most cases, the microorganisms selected to obtain protein are yeasts, due to they contain between 40-80% of true protein in their cells; such as Kluyveromyces marxianus [16]. Table 3 lists some species of yeast grown on different substrates and their respective protein percentages. Candida utilis, Kluyveromyces fragilis and Saccharomyces cerevisiae can be highlighted as the main producers of single-celled protein with production percentages around 50% of its dry weight.

Methodology
Whole whey was used as a growing medium for all fermentations, it was provided by a local cheese shop in the community of Santa María Tonantzintla, Puebla. Whey was characterized by proximal analysis, with the samples being analysed by triplicate.

Protein Determination
Soluble protein concentration was carried out by the micro-Kjeldahl method, modified by NOM-155-SCFI-2003 [17]. 2.5 mL sample was placed in a Kjeldahl tube that was previously added 3 g of potassium sulphate and 0.25 g of pentahydrate copper sulphate, subsequently 5 mL of concentrated sulphuric acid were added. The tubes were placed in the digestion equipment with an initial temperature of 180-230°C for 30 min to avoid foaming, gradually increasing the temperature to 410-430°C; digestion stopped when the solution was clarified. Once the samples are digested, they are kept at rest until they reach room temperature and 20 mL of water and 15 mL of sodium hydroxide are added to 50%. In an Erlenmeyer flask, 50 mL of 2% boric acid and 5 drops of the Shiro Tashiro indicator were placed. Finally, the tube and flask are placed in the distillation unit and the receiving platform, respectively.
Distillation is carried out until 150 mL of solution is recovered in the receiving flask. The distillate was titled with 0.1 N hydrochloric acid and the percentage of proteins was calculated using the formula of Equation 1.
Where: V=Volume spent on titration N=Normality of hydrochloric acid 0.014=Nitrogen milliequivalent 6.38=Nitrogen-protein ratio factor in milk M=Wet sample mass

Lactose
Lactose was quantified using the DNS method, using a 4 g/L lactose standard for the development of a calibration curve and 1:40 dilution of the sample [18]. The standard curve was drawn up according with to lay out in Table 4, which specifies the added volumes of standard solution, water and DNS. The samples were prepared identically to tube 6 of the standard curves, replacing the standard solution with the diluted sample. All tubes were boiled water bath for 5 min, when cooled they were added 1.25 mL of water and the absorbance was read at 540 nm. With the results obtained from the standard solution, a graph was drawn up and a linear regression equation was obtained, in which the result absorbance values of each sample were replaced in the independent variable to calculate the lactose concentrations (dependent variable).

Total Solids
Total solids were determined according to the methodology specified in NMX-F-527-1992 [19]. To that end was necessary to place porcelain capsules at constant weight and weigh them. In them were placed 40 mL sample, they were subjected to water bath in order to evaporate as much water as possible and then dried in stovetop at 110°C to constant weight. Each capsule was cooled in desiccator and weighed.
Equation 2 shows the formula with which the solids content was calculated in the sample.

Humidity
Humidity content was made by weight difference, as indicated in NMX-F-083-1986 [20]. In constant and previously heavy weight porcelain capsules, 40 g of sample was placed and dried at 110°C on the stovetop until constant weight was reached. All capsules were transferred to the desiccator for 1 hour or until they reached room temperature and then weighed. Equation 3 shows how the humidity percentage calculation was performed.
(Pmh − Pms) (100) %Humidity=M Where: Pmh=weight of the capsule with wet sample Pms=Weight of capsule with dry sample M=Wet sample mass

Ashes
The ash content was determined using the methodology described in NMX-F-066-S-1978 [21]. The capsules with dry sample result of the determination of total solids were placed on an electric grill to burn the contents until no smoke was detached. Immediately, they were then placed inside a muffle furnace until the sample was fully calcined (800°C / 30 min). The capsules were allowed to cool inside the muffle furnace, then placed in the desiccator until they reached room temperature and weighed. The calculation of the ash content was performed using Equation 4.

Lactoserum Physicochemical Characterization
The solution was titled with ammonium thiocionate 0.1 N until the appearance of a brick red precipitate. The chloride content is expressed as a percentage of sodium chloride, whose milliequivalent is 0.0585, as expressed in Equation 5.

pH
pH was measured by a Hanna-brand digital potentiometer previously calibrated to pH 4 and 7. The reading was made by direct introduction of the electrode into a beaker with the sample.

Lactic Acid
The percentage of lactic acid was quantified by the determination of acidity by volumetric method as established by the NMX-F-206-1986 standard [20]. In Erlenmeyer flasks were placed 10 g of sample, 100 mL of distilled water and 3 drops of phenolphthalein indicator. The mixture was titled with 0.1 N sodium hydroxide until persistent faint pink is observed for approximately 15 seconds.
The calculation was made using the formula of Equation 6, where, 90 is the equivalent of lactic acid.

Microorganisms
The filamentous fungus Aspergillus oryzae and the yeast Saccharomyces cerevisiae strain ATCC 9763 were used both provided by the Faculty of Chemical Sciences of UNAM. They were planted in potato-dextrose agar plates (PDAs), then in tubes with 10 mL of PDA broth, were incubated at 30°C, 72 hours and 28°C, 48 hours respectively and kept in refrigeration until use.

Lactoserum Pre-treatment
Lactoserum that would be sterilized at 120°C as part of heat treatment, was subjected to a process of prior deproteinization; because when exposed to high temperatures, the threedimensional structure of proteins is deployed as a result of the rupture of hydrogen bridges between polypeptide chains and aggregates are formed that interfere with biomass determinations. The deproteinization was carried out by heating the whey to 100°C for 15 min and cooling sharply in the ice bath, then vacuum filtered with standard filter paper.

Experimental Design
The factorial design used for evaluation of growth of microorganisms in lactoserum was 3 factors and 2 levels, in other words, an experimental design 23. To this end, the following factors were taken into account: pasteurization or sterilization temperature, 60 and 120°C; pH, 4 and 7; yeast extract 0 and 10 g/L, as seen in Table 5.
Each treatment was performed in duplicate and the results obtained were analyzed using a variance analysis (ANOVA) through Minitab 17 statistical software in order to determine the most impact factors and which treatments are significantly different from each other.  For aspergillus oryzae crops each flask with 100 mL of treated lactoserum was inoculated with 0.07 g/L biomass and grown for 72 hours at 30°C. In the case of crops of Saccharomyces cerevisiae each flask with 100 mL of treated lactoserum was inoculated with 0.07 g/L of biomass and grown for 48 hours at 28°C.

SCulture Medium
Lactoserum used was subjected to the conditions of the best treatment according to the results obtained in the previous experiments. These were 60°C temperature (pasteurization), pH of 7.0 and absence of yeast extract for Aspergillus oryzae; in contrast, the culture of Saccharomyces cerevisiae was performed under the same conditions, but adding 10 g/L of yeast extract to the substrate.

Inoculum
The initial inoculum was 10% of the total volume of the medium. It was prepared from test tubes with 10 ml of PDA broth incubated with each microorganism under the temperature and time conditions exposed above. Each tube was then poured into Erlenmeyer flasks with 200 ml of lactoserum for 72 hours, 28°C for Aspergillus oryzae and 48 hours, 30°C for Saccharomyces cerevisiae, both at 100 rpm.

Crop Conditions
A 4 L bioreactor with a working volume of 2 L was used for the batch culture of Aspergillus oryzae. The medium was pasteurized inside the reactor for 20 min at 60°C. The inoculum was poured under aseptic conditions and the cultivation was carried out keeping the temperature at 30°C and aeration of 1 vvm. The cultivation of Saccharomyces cerevisiae was carried out under the same conditions; however, the temperature was adjusted to 28°C.

Inoculum
An inoculum was prepared for each microorganism under the same conditions exposed above, decreasing the volume of the flasks to 100 mL of substrate, so that together they increase a volume equal to 10% of the workload.

Inoculum and Cultivation Conditions
The inocules prepared for this crop were made as mentioned above. Initially, the cultivation of Aspergillus oryzae was carried out in a working volume of 500 mL of whole lactoserum at 30°C for 72 hours. Subsequently 300 mL of the substrate was transferred to the second reactor and inoculated with Saccharomyces cerevisiae. In the first reactor 300 mL of new substrate was added and successive additions were made every 48 hours after the media was removed from the second reactor. Samples were collected every 24 hours at the exit of the first reactor, the filtration system and the second reactor; each sample was determined biomass and sugars.

Biomass Determination
The biomass concentration was determined by gravimetric method. 5 mL was taken from each sample of the S. cerevisae crop were centrifuged at 5,500 rpm for 5 minutes in Falcon tubes at constant weight. Instantly, the supernatant was separated and reserved for further analysis, while the pill was centrifuged twice more with water and dried at 80°C to constant weight. For A. oryzae and mixed crops, 2 mL of each sample were taken, these were centrifuged at 16,000 rpm for 10 minutes in Eppendorf tubes at constant weight. Subsequently, the procedure already described for S. cerevisiae was followed. Once the display has completely dried, they were cooled and weighed. The calculation of the generated biomass was performed using the Equation, expressing itself in g of Biomass/L of substrate.
(Ptm − Ptv) %Biomass=v (7) Where: Ptm=Weight of tube with dry sample Ptv=Weight of the empty tube

Lactose Determination
From the supernatant recovered from each sample, the quantification of residual lactose was performed using the reducing sugar technique; using a dilution factor of 1:25 for all samples from the A. oryzae culture and 1:10 for those corresponding to S. cerevisiae.

Characterization Physicochemical of The Single-Cell Protein
The residual broth of each culture with the biomass included in it was frozen and subsequently freeze-dried in order to obtain a product in the form of a powder on a dry basis and thus be able to quantify the different constituents present in it.

Raw Material Characterization
Lactoserum chemical composition from Santa Maria Tonantzintla is summarized in Table 6 while comparing the results obtained in two other related works from Veracruz, Mexico [22] and Miranda [23]. The lactose content reported in the other works differs slightly with what is determined in this work; however, the results are kept in the same order of magnitude and there is no significant difference. The percentage of total protein is similar to that reported by Miranda [23] although low, compared to that exposed by Hernández [22]. Similar behaviors are shown in pH, humidity, total solids and ash determinations; where, although the results are close, small differences are observed. It should be noted that the chemical composition of the dairy industry's waste depends on a number of factors ranging from feeding and rearing of cattle to handling milk and final product [24].

Cultivation of Aspergillus Oryzae
Once the raw material was characterized, cultivations were made under the conditions specified in the methodology for factorial design. Figure 2 shows the responses (biomass production) obtained in each experiment. In this, it is observed that the treatment with the best response corresponds to treatment 7 of the experimental design (pH=7, T=60, EL=0) followed by treatment 6 (pH=4, T=120, EL=0); however, it is noted that there is not significant difference between two treatments since they share the same letter in the classification resulting from the statistical study. The selection criterion was therefore based on both economic aspects of both time and economic resources. Biomass production is similar in both culture because fungi of the genus Aspergillus have the ability to grow in acid pH as they are producting organisms of organic acids and withstand pH conditions between 4 and 6 [25]; thereby, a value of 0.44% of total protein and 4.85% lactose for deproteinized whey [26] has been reported, i.e. the main source of carbon is not affected by the deproteinization process and therefore does not limit the growth of the micro-organism as long as nitrogen is available, which restricts biomass production by reaching minimum concentrations [25,27].
The criterion to consider in determining treatment 7 as suitable for batch culture was that it allows the use of whole lactoserum without deproteinizing, since pasteurization at 60°C does not require prior deproteinization of the substrate; therefore, it does not require pretreatment or generate waste.
Source: own elaboration.  Table 7 groups the effects of the three factors on biomass production resulting from ANOVA. Those values of P (Probability) lesser than 0.05 represent a significant effect on the response, which in this case represents the biomass produced. Thus, it is observed that the pH and temperature factors have a significant effect on biomass production, but not the addition of yeast extract. In contrast, it is observed that both double and triple interactions do not generate relevant effects on the response. Several authors have studied the influence of different sources of carbon and nitrogen, as well as their relationship, on the production of biomass and metabolites, Torres et al., [28]. report that the carbon source is the main factor involved in the production of fungal biomass when it is in concentrations close to 50 g/L, but not when the sugar concentration is low and nitrogen becomes the limiting element [28].
In that regarde, yeast extract is a source of nitrogen rather than carbon and since lactoserum itself contains organic nitrogen from casein and a mixture of amino acids, this element does not limit the growth of the fungus as long as it exists lactose; therefore, the addition of yeast extract is not a factor that significantly affects biomass production. Changes that occur when each factor changes from its lowest value to its high value were also evaluated; this helps us to know the time when the best production is obtained according to the individual valuation of each factor (Table 7). Table 7 shows that biomass production increases by the lowest temperature value in contrast to the best response when pH is at 7. However, the effect corresponding to yeast extract did not have a significant effect between low and high value; a small increase in the response is seen when 10 g/L is added but is not comparable to the effect on the other factors, which is consistent with that concluded from the ANOVA.

Saccharomyces Cerevisiae Culture
S. cerevisae cultures were carried out under the conditions indicated in the experimental design of section 3.3.3. Results obtained for the production of yeast biomass are shown in Figure 2. Treatment 6 (pH=7, T=60, EL=10) allowed higher biomass production of S. cerevisiae, however, no significant difference is observed with treatment 2 (pH=4, T=120, EL=10) but as it was in case of A. oryzae, this treatment requires the removal of lactoserum protein by heat treatment which generates waste and greater cost of time and effort. In this case a phenomenon similar to that observed in A. oryzae culture is observed in that, even if treatment 2 has no observed protein growth, again it is due to the fact that biomass production initially depends on the carbon source.
Since yeast is unable to break down lactose, its growth depends on the added glucose in the yeast extract. [29]. they performed S. cerevisiae cultures in lactoserum without pH control because it has pH regulation capabilities and yeast resists adverse acidic conditions; therefore, it is able to reproduce even at pH close to 4 [29]. Figure 3 also shows letter classification of treatments that are significantly different from each other, so that treatments that share some letters are the same with each other. It is therefore confirmed that there are no significant differences between treatments 2 and 6 that showed the best responses, which is why the selection of the treatment to be used depended on the economic factors. With the results obtained from the experimental design, ANOVA was carried out to evaluate the effect of each factor on biomass production. Table 8 shows that factors with significant effect on response (P<0.05) are the interaction between temperature-pH and yeast extract-temperature.
Source: own elaboration.  In order to validate the results obtained in this work, a bibliographic review was carried out to make comparisons and obtain some conclusions. Table 9 shows some related works and the contrast of their results with those presented in this paper. It should be noted that in most cases the microorganism, substrate and duration of the culture were different from those used in this research, however, they are close references that allow for a comparative analysis. [25] As well as used Aspergillus niger for whole whey, hydrolyzed and/or deproteinized serum cultures; this filament fungus belongs to the same species of A. oryzae, so similar results would be expected.

Discussion
However, results reported by López et al. [25] are higher than 20 g/L, in cultures whose duration was to 6 days and with previous hydrolysis of lactose that allows to microorganism to concentrate its energy on reproductive functions and not in metabolic functions. A similar situation is presented in the results reported by Leal, in which case, biomass production in whole whey after 8 days is approximately 2 g/L higher than results obtained in this work in 3 days of culturing.
On the other hand, when is compared the volumetric speed of biomass generation, it is observed that Lopez et al. [25] report a slightly higher value for A. niger in deproteinized and hydrolyzed lactoserum than reported in this work; furthermore, Leal et al. [30] report lower productivity values than reported in this work. It should be noted that both references were added with salts and the work volume was smaller than that used for fermentations in this work, so, as noted in the results obtained from the previous treatments, in small volumes the microorganism produces greater amounts of biomass.
Cortés-Sánchez et al. [31] report a production of 12.25 g/L of A. oryzae in fat-free milk after 4 days of cultivation; i.e., it reports twice as much production as reported in this work; however, the work volume was 35 mL. The cultivation of S. cerevisiae made by Pisano et el. [29] was carried out in deproteinized and hydrolyzed lactoserum obtaining a production of 7.8 g/L of biomass in 24 hours, unlike the 6.5 g/L produced in this work after 48 hours. Although the cultivation carried out in this research showed a higher substrate consumption than 90%, the yeast did not grow as expected; however, in this work the substrate treatment performed by Pisano et al was not performed [29]. As regards mixed crops, no references of the S. cerevisiae and A. orizae culture were found. However, Moeni et al [32] cultivated a culture with two types of yeast: S. cerevisiae and Kluyveromyces lactis, the latter is a producer of ∝galactosidase and is able to hydrolyzing lactose. Both yeasts produced 22.38 g/L of biomass in 48 hours growing in deproteinized lactoserum as a substrate, a yield 4 times higher than obtained in this work; it should be noted that no salts were added in this work. Finally, as regards multistage crops, it is observed that in stage 1 the accumulated biomass production was 22.25 g/L while in stage 2 it was 15 g/L; 37.25 g/L in total. This result turned out to be twice as minor as reported by Tellez et al. [33] however, the substrate used was not the same, as shown in Table 9.

Conclusions
The results obtained from lactoserum characterization were similar to those reported by other authors, especially in lactose concentrations (3.63%) protein (0.90%) which are within the ranges reported in the literature. The ANOVA study carried out on the different treatments for the cultivation of A. oryzae and S. cerevisiae showed that the best conditions for the cultivation of both microorganisms in whole whey are: pH=7 and pasteurization at 60°C. Although for yeast case was necessary to add yeast extract to the substrate. Both microorganisms are able to consume the sugars contained in lactoserum and, in turn, produce biomass; obtaining maximum biomass values of 6.5 g/L for axenic cultures of S. cerevisiae and A. oryzae, 4.80 g/L for mixed cultivation, The microorganism that develops best in whole lactoserum is A. oryzae in both axénic and staged cultivation, highlighting the latter for allowing greater productivity. The protein content of final product of the culture of S. cerevisiae (63%), A. oryzae (41%) mixed (32%) obtained is similar to what was reported in the literature (30%-54%).