WO2024108287A1

WO2024108287A1 - Process for producing leaven on industrial scale

Info

Publication number: WO2024108287A1
Application number: PCT/BR2023/050408
Authority: WO
Inventors: Ronaldo BIONDO; Ivo RISCHBIETER
Original assignee: Yeastz Empresa De Techs Para Fermento Ltda
Priority date: 2022-11-24
Filing date: 2023-11-23
Publication date: 2024-05-30

Abstract

The invention refers to a process for producing leaven on industrial scale, comprising the provision of a source of carbon comprising hydrolyzed sucrose, as a carbon source for the aerobic fermentation step to produce leaven. The present invention also refers to a system to perform said process, comprising at least one aerobic fermentation unit for which a source of carbon comprising hydrolyzed source is provided as a carbon source for leaven. In a preferred embodiment of the present invention, sucrose hydrolysis is performed by enzymatic inversion as obtained from leaven produced by the industrial productive chain of leaven. The invention also refers to the use of hydrolyzed sucrose as a source of carbon in the aerobic fermentation step of leaven. The embodiments of the invention allow to increase the rate of conversion of reducing sugars into leaven, as observed at the end of the aerobic fermentation step.

Description

PROCESS FOR PRODUCING LEAVEN ON INDUSTRIAL SCALE

Field of the Invention

The present invention refers to the field of leaven production, preferably yeast. Particularly, the present invention refers to a process for producing leaven on industrial scale, a system to perform said process and the use of a carbon source comprising hydrolyzed sucrose as a source of carbon for the leaven aerobic fermenting step in the process and/or system of the invention. The present invention increases the conversion rate of sucrose reducing sugars into leaven, which can be found at the end of the aerobic fermentation step.

Background of the invention

The first references to the use of yeasts were found in the Caucasus and Mesopotamia regions, dating back to about 7,000 b.C. They were applied in fermentation, aiming to produce alcoholic beverages and baker’s yeast. However, only in the 19^th century, Louis Pasteur isolated and identified yeasts, showing that they were microorganisms which were able to ferment sugar, producing carbon dioxide (CO2) and ethanol (Gomez-Pastor, R., Perez-Torrado, R., Garre, E., Matallana, E. (2011), Recent advances in yeast biomass production, in: D. Matovic (ed.), Biomass - Detection, Production and Usage, pages 201-222; Bekatorou, A., Psarianos, C., Koutinas, A. A. (2006), Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415).

Yeasts are used still today for the same purposes. They have also become considerably more important in the market for use to increase the nutritional value of food products as sources of proteins and vitamins, as well as for their use in enzyme production, with the advent of biotechnology.

The industrial production of yeasts for leaven started in Europe. In 1792, the first pressed baker’s and brewer’s yeasts were produced in England. They were available in northern Europe in 1800. In the U. S. A., a pressed yeast from an improved strain was introduced in 1868, facilitating bread production on large scale.

Despite being relatively easily manipulated in laboratory, the industrial production of any microorganism faces various challenges. There are not only many variants acting sequentially or simultaneously to promote the ideal balance of optimal conditions, allowing the adequate growth of those living beings, but there may also be particularities which are difficult to forecast, when implementing commercial production on large scale.

The industrial process for leaven production, particularly yeast, has been slowly improved with time, with a few technological jumps, such as the inclusion of continued aeration in the fermenting step and the slow introduction of sugar into the reactor, besides process automation. Other studies focused on the modulation of the respiratory and/or fermentation capacity, many of which using molecular biology skills, or the modulation of the yeast’s carbon metabolism. The search for innovation in the leaven production process has also focused on improving the rate of fermentable sugars, concentrating in other sugars than sucrose (Nafajpour, G. D., Shan, C. P., 2003, Enzymatic hydrolysis of molasses, Bioresource Technology 86, 91-94), or also attempting to substitute molasses with alternative glucose sources, like corn glucose. However, this substitution brings disadvantages, since those glucose sources usually do not have all the nutrients, thus requiring nutritional complementation, increasing production process costs (Spigno, G., Fumi, M., De Faveri, D., 2009, Glucose syrup and corn steep liquor as alternative to molasses substrates for production of baking-quality yeast, Chemical Engineering Transactions, 17, 843-848).

As an example, WO 00/61722 discloses a genetically modified yeast, wherein the genes involved in glucose repression (genes HXK2 or their analogues) were deleted, thus enabling the yeast to grow and increase yielding during the respiratory process. WO 98/26079 discloses a yeast which glucose repression is reduced or non-existent, by controlled deregulation of glucose-repressible genes (as a function of the overexpression of a specific transcriptional activator - HAP4 or its analogues - from a glucose-insensitive promoter), so that the yeast thus obtained may have improved respiratory capacity, increasing biomass yielding. EP 1728854 discloses a process for producing yeast biomass on commercial scale, wherein the yeast cells functionally overexpress a transcriptional activator (HAP1), so to improve their respiratory and fermenting capacity. WO 2015/042245 discloses a process to increase yeast culture growth and biomass by adding ethanol during the log growth stage. US 2012/0128853 discloses a method for producing yeast from a substrate constituted by date syrup diluted in molasses. US 2015/259694 discloses a method for producing yeast from new strains of the yeast Saccharomyces cerevisiae which can be multiplied into a substrate comprising at least one pentose (5-carbon sugar - C5), which multiplication rate and speed are appropriate for yeast industrial production.

Therefore, despite yeasts having been industrially produced for many years, the search for better productivity at always lower costs, compatible with a competitive market, and with easy implementation is still required. The productivity increase of the solutions as presented by the state of the art is not easily reached when implemented on industrial scale. It can only be incremental against costly technology and/or bringing technical disadvantages (economical viability) and/or disadvantages for the consumer.

Therefore, the present invention was created, disclosing an improved process, system and use for increasing the conversion rate of sucrose-reducing sugars to produce leaven on industrial scale, in a relatively simple way and without the disadvantages of the solutions proposed by the state of the art. The invention is based on providing, as a carbon source for the aerobic fermentation step to produce leaven, a source of carbon comprising hydrolyzed sucrose, bringing very important gains in said conversion rate in comparison with the traditional skills, currently used by the industry.

Summary of the invention

Therefore, an object of the invention is to provide a process for producing leaven on industrial scale, comprising the provision, as a carbon source at the aerobic fermentation step to produce leaven, of a source of carbon comprising hydrolyzed sucrose. In a preferred embodiment of the process of the invention, sucrose hydrolysis is performed by enzymatic inversion, chemical inversion and/or resins.

In another preferred embodiment of the process of the invention, enzymatic inversion is performed by invertase, which may be originated from outside the industrial productive chain, such as of commercial origin and/or specifically produced as a provider for the productive chain at issue, and/or obtained from the leaven produced by the industrial productive chain itself.

In still another preferred embodiment of the process of the invention, chemical inversion is performed with one or more acids, wherein such one or more acids is/are selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, citric acid and lactic acid.

In one more preferred embodiment of the process of the invention, the process comprises the steps of: a. preparing the starting leaven inoculate; b. propagating the leaven prepared by (a); c. promoting the aerobic fermentation of the leaven prepared by (b) to produce leaven; wherein a source of carbon comprising hydrolyzed sucrose is provided as the carbon source; d. separating and/or purifying the product prepared by (c) to concentrate the leaven; and e. filtering, filtering and pressing or filtering, pressing and drying the product obtained in (d).

In still another preferred embodiment of the process of the invention, leaven is yeast, more preferably of the genus Saccharomyces spp., even more preferably from the species Saccharomyces cerevisiae.

In still another preferred embodiment of the process of the invention, sucrose is included in the carbon source used for fermentation, wherein said source is selected from the group consisting of granulated sugar, pure sucrose, VHP (Very High Polarization) sugar, VVHP (Very Very High Polarization) sugar, demerara sugar, brown sugar, concentrated sugar cane juice, molasses and must, or any of their combinations, said sources being preferably produced from sugar cane or sugar beet.

In still another preferred embodiment of the process of the invention, between 50 and 100% sucrose is hydrolyzed, preferably between 85 and 99.9%, more preferably between 90 and 99.9%.

In still another preferred embodiment of the process of the invention, aerobic fermentation is performed under air supply between 0.1 and 2.5 wm, temperature between 28.5 °C and 34 °C and pH between 4.0 and 5.5.

In still another embodiment of the process of the invention, sucrose as a carbon source has above 1 °Brix, preferably between 1 and 99 °Brix, more preferably between 15 and 80 °Brix.

In still another preferred embodiment of the process of the invention, the leaven production process increases ART conversion into leaven at the end of the aerobic fermentation step.

A second object of the invention refers to a system to perform the process for producing leaven on industrial scale of the invention, comprising at least one aerobic fermentation unit (2) for which a source of carbon comprising hydrolyzed sucrose is provided as a carbon source for leaven production.

In a preferred embodiment of the system of the invention, the system also comprises at least one unit to produce invertase enzyme (41 , 42), which may (41) or not (42) be connected to the industrial productive chain.

In another preferred embodiment of the system of the invention, the system also comprises at least one storage and/or treatment unit for the carbon source (5) comprising sucrose, to which the invertase enzyme is provided, where the hydrolysis of the sucrose to be used as a carbon source for the aerobic fermentation unit (2) to produce leaven will take place.

In still another preferred embodiment of the system of the invention, the system also comprises: at least one leaven propagation unit (1), to receive the previously prepared leaven from the initial inoculate; at least one separation and/or purification unit (3); and/or at least one filtering, filtering and pressing or filtering, pressing and drying unit (6).

In still another preferred embodiment of the system of the invention, at least one leaven propagation unit (1) is connected to at least one aerobic fermentation unit (2) which, on the other hand, is connected to at least one carbon source storage and/or treatment unit (5) and to at least one separation and/or purification unit (3), which is connected to at least one filtering, filtering and pressing or filtering, pressing and drying unit (6).

In still another preferred embodiment of the system of the invention, sucrose included in the carbon source of at least one storage and/or treatment unit (5) is hydrolyzed by the invertase enzyme received from at least one invertase enzyme production unit (41 , 42), which is preferably connected to the industrial productive chain (41), wherein a carbon source comprising hydrolyzed sucrose from at least one storage and/or treatment unit (5) is then used as a carbon source for leaven in at least one aerobic fermentation unit (2) for producing leaven, so to generate leaven biomass, which is separated and/or purified in the at least one separation and/or purification unit (3), and filtered, filtered and pressed, or filtered, pressed and dried in the at least one filtering, filtering and pressing or filtering, pressing and drying unit (6); wherein the leaven of at least one aerobic fermentation unit (2) was previously propagated in at least one leaven propagation unit (1), after receiving the previously prepared leaven from the initial inoculate.

A third object of the invention refers to the use of a source of carbon comprising hydrolyzed sucrose as a carbon source for the aerobic fermentation step of leaven in the process for producing leaven on industrial scale of the invention, and/or in the system to perform the process for producing leaven on industrial scale of the invention.

Brief description of figures

The invention may also be understood based on the details presented by the Figures.

Figure 1 shows the schematic representation of the sucrose metabolism by S. cerevisiae. Sucrose may be externally hydrolyzed by an external invertase produced by yeast and internalized as glucose and fructose by the hexose transporters Hxt, or sucrose may be transported by Agt1 or MALT permeases, hydrolyzed by a cytoplasmic invertase and consumed by cell metabolism.

Figure 2 shows the schematic representation of glucose metabolism into yeast. 1 - glucose transportation: facilitated diffusion or active transportation; 2 - glucogen and tre-halose formation; 3 - glycolysis; 4 - pyruvate decarboxylase; 5 - dehydrogenase alcohol; 6 - dehydrogenase acetaldehyde; 7 - acetyl-CoA synthase; 8 - acetil-CoA transporter; 9 - acetate transportation to mitochondria; 10 - piruvate transportation to mitochondria; 11 - pyruvate dehydrogenase; 12 - Krebs cycle; 13 - pyruvate carboxylase; 14 - ADP/ATP translocator and 15 - ATP formation via oxidative phosphorylation.

Figure 3 shows the schematic representation of glucose gene repression in yeast. An intricate metabolic communication network regulates gene expression in the cell core, by adapting the cell structure to the conditions as indicated by the external medium. The presence of glucose induces repressors for proteincoding genes for the stress response chain, including genes involved in gluconeogenesis, breathing, picking and breaking alternative carbon sources, such as maltose (MAL genes) and galactose (GAL genes), also acting at the expression levels of enzymes such as invertase.

Figure 4 shows a scheme of the process or system for producing leaven from molasses (the scheme was adapted from Bekatorou, A., Psarianos, C., Koutinas, A. A. (2006), Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415).

Figure 5 shows a set of preferred embodiments of the process and system of the invention, showing, among others, the production unit for the invertase enzyme obtained from leaven produced by the industrial productive chain for leaven (41) (“Enzyme”), as well as the production unit for the invertase enzyme originated outside the industrial productive chain (42) (“Commercial Enzyme”) for leaven. Said embodiments may be alternatively or simultaneously included.

Figure 6 shows the experimental result representing the enzymatic inversion rate (% inversion X time) in molasses and must (Example 1).

Figure 7 shows the experimental result representing the biomass (wet mass) growth performance for Fleischmann “salty mass” leaven by the time of cultivation from gross or inverted must (Example 1).

Figure 8 shows the experimental result representing the biomass (wet mass) growth performance for Fleischmann “salty mass” leaven by the time of cultivation from gross or inverted must (Example 1).

Figure 9 shows the experimental result representing the biomass (wet mass) growth performance for Fleischmann “sweet mass” leaven by the time of cultivation from gross or inverted must (Example 1).

Figures 10 and 11 show the experimental results for the biomass (wet mass) growth performance for Fleischmann “sweet mass” leaven by the time of cultivation from gross or inverted must on industrial scale (Example 2).

Detailed description of the invention

The invention refers to a process for producing leaven on industrial scale, comprising the provision, as a carbon source at the aerobic fermentation step to produce leaven, of a source of carbon comprising hydrolyzed sucrose. The invention also refers to a system to perform said process, comprising at least one aerobic fermentation unit to which, as the carbon source for leaven, a source of carbon comprising hydrolyzed sucrose is provided. Finally, the invention also refers to the use of a source of carbon comprising hydrolyzed sucrose as a carbon source for the leaven aerobic fermentation step in the process and/or system of the invention. The invention allows to increase the conversion rate of sucrose reducing sugars into leaven, which is observed at the end of the aerobic fermentation step.

Throughout this document, unless indicated otherwise, the limits of a range of values are included within that range. Also, the ranges of values include all integers and fractions enclosed within said ranges.

Throughout this document, the singular forms “a”, “an” and “the” include the singular, their respective plurals and vice-versa, unless the context clearly indicates otherwise.

Throughout this document, the term “at least one” is equivalent to the term “one or more”, and they mean one or more members, or at least one member of a group of members. These terms include any one from > 1 , > 2, > 3, > 4, > 5, > 6, > 7, etc. of said members and even all said members.

Throughout this document, the terms “about” and “approximately”, when referring to a measurable amount as a parameter, a quantity, a temporary period and similar, mean variations of the specified value, such as +/- 10% variations or less, preferably +/- 5% or less, more preferably +/- 1 % or less and even more preferably +/- 0.1 % or less, as long as reasonable, while these variations are adequate for each appropriate parameter of the invention. It should be understood that the amount referred to by the term “about" is also specifically and preferably disclosed.

Throughout this document, terms and expressions such as “preferably”, “particularly”, “as an example”, “like”, “such as”, “more particularly”, “more preferably” and similar, as well as their variations, should be understood as fully optional characteristics, preferred embodiments or possible and non-exhaustive examples, not limiting the scope of the invention. Throughout this document, the word “comprises” and any variations such as “comprise” or “comprising” should be interpreted as “open terms”, and may cause the inclusion of additional elements or groups of elements, which have not been explicitly disclosed and do not have limitative character. The terms “including”, “covering” and any of their variations are used in the same fashion.

Throughout this document, the word “consists” and any variations such as “consist" or “consisting” should be interpreted as “closed terms”, and should not indicate the inclusion of additional elements or groups of elements which have not been explicitly disclosed, since they have limitative character.

When not explicitly indicated otherwise, all acronyms, technical terms and/or expressions should be understood as having their generally used meanings, as widely known in the technical field of the invention. In some cases, the terms with commonly understood meanings are defined by this document to bring clarity and/or for prompt reference, and the inclusion of said definitions in this document should not be necessarily understood as representing substantial differences over the usual understanding of the state of the art.

If not explicitly indicated otherwise, the skills and procedures disclosed or indicated by this document are usually well understood and employed by using conventional methods, based on the available literature and the knowledge of an expert in the art without undue experimentation.

Throughout this document, all titles and subtitles are only used for convenience and should not be interpreted as limitations of the present invention.

Throughout this document, all the mentioned references, books, articles, patent documents and others should be considered as being incorporated as reference.

Throughout this document, the term “leaven” refers to biological, commercial, natural (selected from nature) or genetically modified leaven, capable of performing aerobic and/or anaerobic fermentation of organic matter. The best known leaven is yeast, or “baker’s yeast”, particularly from the genus Saccharomyces spp, such as Saccharomyces cerevisiae. In a few contexts throughout this document, the word leaven may be included in the term “leaven biomass” or simply “biomass”. Said term and said word should be interpreted as a synonym of “leaven”, but may be also understood as the microorganism as successfully propagated.

In the present description, the term “industrial scale” should be understood as a synonym of “commercial scale”. This means that this term indicates its full and successful implementation or implementation ability for industrial production intended for commercialization, on small or large scale, in opposition to the embodiments in laboratory environments or which are merely experimental.

In this document, the term “increase in leaven production”, “rate increase”, “factor increase” or simply “increase in ART conversion into leaven” refers to any increase over the increase as observed for a group, such as a control group, as well as any increase of the expected production, e. g. by conventional leaven production skills, or reported in the state of the art by any leaven production skills.

The present invention refers to a process for producing leaven on industrial scale, comprising the provision of a source of carbon comprising hydrolyzed sucrose as a carbon source for the aerobic fermentation step to produce leaven. Sucrose hydrolysis of the invention may be performed by any method as known in the state of the art, such as by enzymatic inversion, chemical inversion and/or by resins, with enzymatic inversion being preferred. Said enzymatic inversion is preferably performed by invertase, which origin may be from outside the industrial productive chain, e. g. from commercial origin and/or specifically produced for supply to the productive chain at issue, and/or obtained from the leaven produced by the industrial productive chain itself. Therefore, we should highlight that the invertase obtained from the leaven produced by the industrial productive chain itself, as disclosed by the invention, may be economically favorable, since it makes use of a part of the product obtained from the production process itself to generate raw materials to be used for the same production chain, so to obtain additional quantities of leaven.

The sucrose inversion process, preferably performed in a full mixture reactor, takes place at appropriate temperature and pH for the invertase enzyme to act (between 50 and 60 °C, preferably 55 °C, and pH between 4 and 5, preferably pH 4.5). Sucrose as included in the carbon source raw material, such as honey, molasses and others, may be inverted under various sucrose concentrations, and, after the addition of the invertase enzyme (or enzyme complex, as detailed below), the process starts and the total period for the desired inversion rate depends on the amount of sucrose, the activity/quantity of the enzyme and process conditions. An expert in the art is able to adjust the process accordingly, with no undue experimentation. Preferably, the inverted sucrose follows to the must preparation process, followed by aerobic fermentation, continued or in batches, feeding the must continuously or intermittently.

Therefore, in a preferred embodiment of the invention, enzymatic inversion by invertase, as obtained from the leaven produced by the industrial productive chain itself, is advantageously proposed. This may be performed by using the processes to obtain invertase (or processes for producing a crude extract of the enzymatic complex, which may be conventional and organic) as disclosed by the document BR 10 2018 010441 1 , incorporated to the present description as reference. According to that document, the processes for obtaining invertase as disclosed comprise the following steps: (i) diluting the fresh leaven; (ii) performing cell lysis; (iii) polishing with salts; (iv) concentrating the resulting leaven; and (v) diluting it in the desired buffer.

In case of chemical inversion, this may be similarly performed by any method disclosed in the state of the art for that purpose. Usually, sucrose syrup is acidified at pH between 2.0 and 2.5 and heated between about 75 °C and 95 °C for about 2 to 3 hours to reach an inversion rate between 60 and 70% of the sucrose present in the syrup. Generally speaking, it is performed with one or more acids, wherein that one or more acids is/are selected from the group consisting of: hydrochloric acid, phosphoric acid, sulfuric acid, citric acid and lactic acid. Methods for performing the chemical inversion of sucrose with acids are well known and may have their parameters adjusted, depending on the acid at issue, the quantity of sucrose to be inverted, etc.

In case of inversion with resin, such as cationic resin, it may also be performed by any method disclosed for that purpose by the state of the art, not being particularly relevant for the present invention (Sinha, C., Gehlawat, 1995, Inversion of sucrose with cation resins, Indian Journal of Chemical Technology 2, 171- 172; Alexandratos, S. D., 2009, Ion-Exchange Resins: A Retrospective from Industrial and Engineering Chemistry, Ind. Eng. Chem. Res. 48, 388-398; Buttersack, C., Hofmann, J., Roger Glaser, R., 2021 , Hydrolysis of Sucrose over Sulfonic Acid Resins Chem. Cat. Chem. 13, 3443-3460).

This is because the process for producing leaven on industrial scale is based on the disclosure by the present invention that the supply of the carbon source comprising hydrolyzed sucrose in the leaven aerobic fermentation step causes a very considerable increase in the rate of conversion of sucrose reducing sugars into leaven, which could not be reasonably expected from the knowledge of the state of the art. Therefore, changes to other steps of the process are accepted and bring the technical characteristic of increased conversion into leaven as disclosed by the present description, not escaping from the scope of the invention.

General processes for obtaining leaven involve at least the following steps: (a) preparing the initial leaven inoculate; (b) propagating the leaven as prepared in (a); (c) promoting the aerobic fermentation of leaven as prepared in (b) to produce leaven, wherein a carbon source is used for fermentation; (d) separating and/or purifying the product as obtained in (c) to concentrate the leaven; and (e) filtering, filtering and pressing or filtering, pressing and drying the product as obtained in (d). An example of a usual process or system of the state of the art is shown on Figure 4. According to the process of the present invention, step (c) provides, as a source of carbon for leaven, a carbon source comprising hydrolyzed sucrose (preferred embodiments are shown on Figure 5).

Using Figure 4 as an example representing a general process for producing leaven of the state of the art, in additional complementation to the steps as disclosed above, yeast strains kept in a laboratory and reactivated for industrial processes are generally used for step (a) preparing the initial leaven inoculate. The continued integrity of the strain is guaranteed by the biochemical and microbiochemical analyses as usually performed. The yeast strain is cultivated in a laboratory with a sterile nutritive solution under controlled conditions, and subsequently used for larger fermentation as pure inoculate. For step (b) propagating the leaven as prepared in (a), a seed tank is generally used. Yeast grown in laboratory is used as a pre-inoculate and starts to be cultivated to obtain the biomass as required for its use on industrial scale. Pure yeasts cells cultivated in an adequately adjusted mixture, usually of molasses, in a laboratory are aseptically transferred to one or more bioreactors “Seed”. For step (c) promoting aerobic fermentation of leaven as prepared in (b) to produce leaven, wherein a carbon source is used for fermentation, an industrial scale reactor is generally used. The inoculate of the seed tank is sent to the industrial reactor, usually representing 10% of the volume of the production reactor, and the production process is conducted by injecting pre-filtered air in proportional volume to the fermented matter and kept until reaching maximum conversion of the substrate into biomass. Yeast cells may be cultivated in a series of fermentation bioreactors, which are operated under aerobic conditions to promote yeast growth. For step (d) separating and/or purifying the product as obtained in (c) to concentrate the leaven, a centrifuge is usually employed. Finally, the step (e) filtering, filtering and pressing, or filtering, pressing and drying the product as obtained in (d) is performed by usual equipments employed for that purpose. Yeast may be sold as a cream, pressed or dehydrated, depending on the final product to be produced (e. g. fresh, dry or instantaneous leaven) and may be then packed and stored under appropriate conditions.

Fresh leaven usually consists of between about 30% and 33% of dry matter, constituted in the following proportion, in average: between 6.5% and 9.3% nitrogen; between 40% and 58% protein; between 35% and 45% carbohydrates; between 4 and 6% lipids; between 5 and 7.5% minerals and various amounts of vitamins, depending on its type and growing conditions. To reach said composition, raw materials rich in minerals and carbon source are used. About 80% of the yeasts commercialized in the world are in cream form (20% dry weight) or pressed yeast (30% dry weight) (Attfield, P. V., 1997, Stress tolerance: the key to effective strains of industrial baker’s yeast, Nature Biotechnology, 15, 1351 ; Bejatiriym, A. et al, 2006, Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415; Chen, S. L., Chiger, M. 1985. Production of baker’s yeast, in: Blanch, H. W., Drew, S., Wang, D. I. C. (eds), Compreensive Biotechnology, pages 429-462, Pergamon Press, NY).

Concerning the process for producing leaven of the present invention, the carbon source as used for fermentation is a carbon source comprising hydrolyzed sucrose, as already indicated. Figure 5 shows preferred embodiments of said process. In a preferred embodiment of the invention, as shown by Figure 5, both molasses, e. g. extracted from sugar cane, included in a tank (52) which is a part of the storage and/or treatment unit for carbon sources (5), and the must obtained and included in another tank (53) of the same unit (5), after a usual pasteurization process, are treated with invertase from the productive chain itself, e. g. by the already disclosed methods. In this case, part of the leaven obtained after step (d) - separating and/or purifying the product obtained in (c) to concentrate the leaven - is directed to a production unit for invertase enzyme (41), wherein the steps to obtain invertase take place, such as disclosed by the document BR 10 2018 010441 1 , incorporated herein as reference. In another preferred embodiment of the invention, commercial invertase is added from the production unit for invertase enzyme of external origin to the productive chain (42), as well as to said molasses (52) and must tanks (53). Enzyme originating from outside the chain could also be used, but specifically produced to supply the productive chain at issue. In one more preferred embodiment, any two or even the three routes (enzyme obtained from the leaven of the productive process itself and/or a commercial, specifically produced enzyme or from an external origin) could be used in the same process. The present invention also includes the treatment of just one of the tanks comprising carbon sources, molasses or must, with invertase. Preferably, the treatment with invertase enzyme is performed in the tank comprising molasses (52) and the must (53) as carbon sources.

As for other possible variations in the general process for producing leaven, these are well known in the state of the art and may be of two kinds: (i) continuous and (ii) semi-discontinuous or fed-batch (Burrows, S., 1970, Baker’s yeast, in The Yeasts, Academic Press, New York; 3 (349-320); Chen, S. L., Chiger, M., 1985, Production of baker’s yeast in comprehensive biotechnology, Pergamon Press; 1^st ed.; 3 (429-461); Roseiro, C. I. S., Baptista, M. S. C. P., 2012, Dimensionamento de uma unidade de produgao de leveduras para a panificagao, Master Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar).

In terms of process, leaven production is mainly performed by means of fed batches of molasses in bioreactors with more than 100 m³. Feeding should be proportional to the exponential growth of the biomass, controlling oxygen transference so to direct yeast metabolism to the production of biomass rather than ethanol. In the final stage of the process, nitrogen source feeding and, later, the feeding of molasses are gradually reduced to zero, aiming to guarantee yeast maturation and the creation of sufficient supply of carbohydrates (glycogen and tre-halose) for storage. The full time of the process is between about 12 h and 14 h, after which the cells are collected and treated for commercial sale, fresh or dehydrated. Typical industrial fermentation has volume productivity between about 2.5 and 3.0 kg/m³/h (Chen, S. L.; Chiger, M., 1985, Production of baker’s yeast in comprehensive biotechnology, Pergamon Press; 1^st ed.; 3 (429-461); George, S., Larsson, G., Olsson, K., Enfors, S. O., 1998, Comparison of the Baker's yeast process performance in laboratory and production scale, Bioprocess Engineering 18, 135- 142; Kristiansen, B. (ed.), Integrated design of a fermentation plant - the production of baker’s yeast, VCH, Weinheim, 1993; Roseiro, C. I. S., Baptista, M. S. C. P., 2012, Dimensionamento de uma unidade de produgao de leveduras para a panificagao, Master Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar). In the present invention, the semi-discontinuous or fed-batch process is preferred.

The most widely used microorganism for leaven production process is the baker’s yeast of genus Saccharomyces, a single-cell fungus which is distinguished from bacteria, among other reasons, for having larger measurements, within the range between 5 and 8 pm diameter, and for associating various intrinsic attributes to the conduction of cell multiplication and generation of biomass, among which its cell multiplication ability in the presence of oxygen, tolerance to temperature and pH variations and osmotolerance [Kavscek, M., Strazar, M., Curk, T. et al, 2015, Yeast as a cell factory: current state and perspectives, Microb. Cell Fact. 14: 94; Steensels, J., Snoek, T., Meersman, E. et al, 2014, Improving industrial yeast strains:

Exploiting natural and artificial diversity, FEMS Microbiol. Rev. 38: 947-995; Tanguler, H., Erten, H., 2008, Utilisation of spent brewer's yeast for yeast extract production by autolysis: The effect of temperature, Food and Bioproducts Processing 86 (4): 317-321 ; Roseiro, C. I. S., Baptista, M. S. C. P. 2012, Dimensionamento de uma unidade de produgao de leveduras para a panificagao, Master Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar; Trevizam, C. J., Molena, C., Dicesar Correia, D., 2013, Estudo dos principals parametros de controle e a agao da trealose no processo de produgao de levedura de panificagao, Revista Engenho, vol. 8).

Yeasts are optional anaerobic microorganisms and may grow in the presence or lack of oxygen. In the presence of oxygen, they convert sugars into CO2, energy and biomass. Under anaerobic conditions, such as alcoholic fermentation, yeasts generally do not grow efficiently, and sugars are converted into products such as ethanol, glycerol and CO2. The main source of carbon and energy for most yeasts is glucose, which is converted by glycolytic route into pyruvate and, by means of the Krebs Cycle, generating energy in the form of adenosine triphosphate (ATP) (Bekatorou, A., Psarianos, C., Koutinas, A. A. (2006), Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415; Amorim, H. V., Leao, R. M. (2005), Fermentagao alcoolica: ciencia e tecnologia, Piracicaba, Brazil: Fermentec, 448p). According to the present invention, the preferred leaven is yeast, more preferably Saccharomyces spp., even more preferably Saccharomyces cerevisiae.

The raw materials used as substrates for the production of yeast biomass are usually agricultural, forest and subproducts from the food industry. There are essentially two kinds of raw materials, depending on the cultivated microorganism: (i) conventional materials, such as starch, molasses, milk serum, fruit and vegetable residues, wood, straw etc.; (ii) non-conventional materials, such as oil derivatives, natural gas, glycerin, ethanol and methanol (Carbonetto, B., Ramsayer, J., Nidelet, T. et al, 2018, Bakery yeasts, a new model for studies in ecology and evolution, Yeast 35: 591-603; Duan, S. F., Han, P. J., Wang, Q. M. et al, 2018, The origin and adaptive evolution of domesticated populations of yeast from Far East Asia, Nat. Commun. 9: 2690; Heitmann, M., Zannini, E., Arendt, E., 2018, Impact of Saccharomyces cerevisiae metabolites produced during fermentation on bread quality parameters: A review, Crit. Rev. Food. Sci. Nutr. 58: 1152-1164; Jay, J. M., Modern Food Microbiology, Chapman & Hall, New York, USA, 1996; Joseph, R., Bachhawat, A. K., 2014, Yeasts: Production and Commercial Uses, in: Batt, C. A., Nielsen, J. (2019), Yeast systems biology: model organism and cell factory, Biotechnol, J. 14: e1800421 ; Tortorello, M. L., Encyclopedia of Food Microbiology, 2 Eds., Oxford: Academic Press, 823-830; Trivedi, N. B., Jacobson, G., Tesch, W. 1986, Baker’s Yeast, Critical Reviews in Biotechnology, 24: 1 , 75-109).

Conventional raw materials also include sugar cane molasses or sugar beet molasses, which are the main substrates used by industrial factories for yeast production for leaven. These materials have been selected for various main reasons, such as: (i) yeasts grow very well by using sugars present in the molasses; (ii) they are economically interesting for being residual products from sugar refineries, which usually send those molasses for alcohol production; and (iii) they contemplate the nutritional requirements of the yeast as a source of carbon, minerals, vitamins, essential elements and organic nitrogen (Bekatorou, A. et al, 2006, Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415; Gomez- Pastor, R., Perez-Torrado, R., Garre, Elena, G., Matall, E., Recent Advances in Yeast Biomass Production, 2011 , DOI: 10.5772/19458; Roseiro, C. I. S., Baptista, M. S. C. P. 2012, Dimensionamento de uma unidade de produgao de leveduras para a panificagao, Master Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar).

The characteristics of sugar cane molasses produced in Brazil may depend on their region and on the extraction process in each sugar mill. Molasses usually have a range between 80 and 90 °Brix (referring to the quantity of soluble solids (sugar) in grams per 100 grams of solution) and these variations are taken into account to guarantee the time of storage of the raw material. The Brix value is also directly related to the Total Reducing Sugars (ART) as present in the molasses. The ART range usually found in Brazilian molasses is between 55% and 65%, including sucrose, glucose, fructose, raffinose, mellibiose and galactose. According to the process for producing leaven on industrial scale of the invention, sucrose has above 1 °Brix, preferably between 1 and 99 °Brix, more preferably between 15 and 80 °Brix.

All these sugars are used by the yeast as a carbon source, preferably glucose, fructose and sucrose, respectively. Generally speaking, baker’s yeast producers manage a stock mix and standardize its use in the process so to obtain the ideal use of sugars in the process. Theoretically, the higher the ART value in the molasses, the lower is the quantity of molasses to be used for fermenting, and consequently, the quantity of molasses used per ton of produced yeast is reduced. However, the main characteristic observed in adequate molasses for the production of baker’s yeast is the ratio from the variation of total reducing sugars to the concentration of the other substances included in the molasses. This ratio is called molasse balance, and may be corrected by adding any other substances (vitamins, minerals, nitrogen and phosphorous) (Bekatorou, A., Psarianos, C., Koutinas, A. A. (2006), Food Grade Yeasts, Food Technol. Biotechnol. 44 (3) 407-415; Gomez-Pastor, R., Perez-Torrado, R., Garre, Elena, G., Matall, E., Recent Advances in Yeast Biomass Production, 2011 , DOI: 10.5772/19458; Trevizam, C. J., Molena, C., Dicesar Correia, D., 2013, Estudo dos principals parametros de controle e a aqao da trealose no processo de produqao de levedura de panificaqao, Revista Engenho, vol. 8).

The physical and chemical standards of sugar cane molasses are usually considered, since they constitute the main raw material of the fermentation process. Therefore, the step of clarifying the molasses is usually relevant for leaven production, avoiding contaminations between leaven and the mud from the molasses, causing adverse effects to fermentation, fermentation color and product durability. A few methods may be used to clarify the molasses, from calcium phosphate precipitation by adding phosphoric acid to the consequent use of a physical separation method, e. g. using centrifuges and filters. There are two forms to treat molasses: clarifying and then sterilizing or first sterilizing and then clarifying. The option to first clarifying and then sterilizing is preferable, considering that, when the process is effected otherwise (first sterilizing and then clarifying), it is possible to remove caramelized products during sterilization, with higher contamination probabilities (Reed, G., Nagodawithana, W. T., Yeast Technology, Van Nostrand Reinhold, 2^nd ed., 1991 ; Roseiro, C. I. S., Baptista, M. S. C. P. 2012, Dimensionamento de uma unidade de produqao de leveduras para a panificaqao, Master Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar; Trevizam, C. J., Molena, C., Dicesar Correia, D., 2013, Estudo dos principals parametros de controle e a aqao da trealose no processo de produqao de levedura de panificaqao, Revista Engenho, vol. 8; White J., Yeast Technology, John Wiley and Sons Inc., 1^st ed., New York, 1954).

According to the present invention, sucrose is present in the carbon source used for fermentation, and said source may be from any origin, most usually from sugar cane and sugar beets, preferably sugar cane. From said sources, it is possible to prepare numerous raw materials, which may be used as a source of sucrose within the present invention. Preferably, the source of sucrose is selected from the group consisting of granulated sugar, pure sucrose, VHP (Very High Polarization) sugar, VVHP (Very Very High Polarization) sugar, demerara sugar, brown sugar, concentrated sugar cane juice, molasses and must, or any of their combinations. According to the process for producing leaven on industrial scale of the invention, most of the sucrose (above 50%), preferably almost all sucrose, is hydrolyzed (inversion above 99% ART of the carbon source, such as molasses/must); particularly between 80 and 99.99% sucrose is hydrolyzed, preferably between 85 and 99.9%, more preferably between 90 and 99.9%.

The use of sugars (widely speaking) by yeasts initially involves their transport to inside the cell. The barrier between the outer and the inner side of the yeast cell consists of the cell wall, the plasma membrane and the periplasmic space. The transport through the plasma membrane is essential to maintain life, for communication between the cells and for the adaptation to environmental changes. Sugars do not freely permeate into biological membranes and the cell absorption of sugars requires the action of transporter proteins. Sugar transporters specifically link their substrate sugar, and then take it to within the yeast cell. Some sugar transporters are highly specific, while others have a wide range of substrates. Sugar transporters mediate two kinds of transport processes within yeast cells: facilitated diffusion with no use of energy and energy-dependent transport via a proton mechanism. These systems are responsible for the excretion of products from the metabolic routes and harmful substances, regulating the ion flow which may be kept under very different intracellular concentrations from those of the external environment. Molecules such as carbon dioxide (CO2), oxygen (O2) and ethanol may cross the membrane by simple diffusion. Furthermore, the diffusion mediated by transporters and channels is facilitated, allowing the solute to go from one side of the membrane to the other, without using metabolic energy and facilitating a concentration gradient (Bisson, L. F. et al, 1993, Yeast Sugar Transporters, Critical Reviews in Biochemistry and Molecular Biology, vol. 28, n° 4, pages 259-308; Pao, S. S. et al, 1998, Major facilitator superfamily, Microbiology and molecular biology reviews, vol. 62, n° 1 , pages 1-34; Saier, M. H., 2000, Vectorial Metabolism and the Evolution of Transport Systems, Journal of Bacteriology, vol. 182, n° 18, pages 5029-5035).

Generally speaking, yeasts may use a wide variety of sugars, but, when various sugars are simultaneously present, the yeast usually consumes them sequentially. Easily assimilated sugars, i. e. monosaccharides, are used in first place. Both glucose and fructose are carried to the yeast cell by members of the hexose transporter family (Hxt), consisting of energy-free facilitated diffusion transporters. Glucose and fructose are absorbed in an initial fermentation stage. Hxt transporters are more efficient for glucose than for fructose. Therefore, glucose is carried more quickly than fructose, even when the initial fructose content is higher. Glucose is the yeast’s preferred substrate over all other carbon hydrates and, in the presence of e. g. maltose and maltotriose, the consumption of these carbon sources is delayed. The most important mechanisms by which glucose causes this delay in the consumption of other sugars are catabolic repression and catabolic inhibition (D’amore, T., Russel, I., Stewart, G. G., 1989, Sugar utilization by yeast during fermentation, Journal of Industrial Microbiology, vol. 4, n° 4, pages 315-323; Meneses, F. J., Henschke, P. A., Jiranek, V., 2002, A Survey of Industrial Strains of Saccharomyces cerevisiae Reveals Numerous Altered Patterns of Maltose and Sucrose Utilisation, Journal of the Institute of Brewing, vol. 108, n° 3, pages 310-321 ; Wieczorke, R. et al, 1999, Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae, FEBS Letters, vol. 464, n° 3, pages 123-128). Direct sucrose uptake analysis by Saccharomyces cerevisiae disclosed the existence of a sucrose cotransporter which is mediated by permease known as Agt1 , besides the one performed by maltose permeases (MALT). Therefore, the strains do not need to hydrolyze sucrose outside the cell to metabolize it, i. e. sucrose is carried to within the cell by means of permeases, where it is hydrolyzed by the intracellular invertase, releasing glucose and fructose molecules which will be later metabolized by the glycolytic pathway. Thus, two routes of use of sucrose are known for the yeasts of the species S. cerevisiae-. (i) by action of extracellular invertase, sucrose is hydrolyzed into glucose and fructose, and their hydrolysis products are carried to within the cell via hexose transporters; or (ii) alternatively, by active transportation, wherein sucrose may be directly uptaken through co-transportation, followed by intracellular hydrolysis (Figure 1) (Barford, J. P., Phillips, P. J., Orlowski, J. H., 1992, A new model of uptake of multiple sugars by S. cerevisiae, Bioprocess Engineering, vol. 7, n° 7, pages 297-302; Santos, E. et al, 1982. Uptake of sucrose by Saccharomyces cerevisiae, Archives of Biochemistry and Biophysics, vol. 216, n° 2, pages 652-660; Stambuk, B. U., Araujo, P. S., 2001 , Kinetics of active alphaglucoside transport in Saccharomyces cerevisiae, FEMS Yeast Research, vol. 1 , n° 1 , pages 73-78).

Generally speaking, when the yeast grows in an aerobic environment, the process is called breathing and, in case of an anaerobic environment, the process is called fermentation. During fermentation, each glucose molecule produces 2 ATPs, while breathing produces 38 ATP molecules. Therefore, to produce the same quantity of ATP, about 18x more glucose should be consumed under anaerobic conditions, in comparison with aerobic conditions. During glycolysis, the glucose molecule is submitted to a series of enzymatic reactions to pyruvate. At that point, in the lack of oxygen, there is the action of the enzyme pyruvate decarboxylase and alcohol dehydrogenase, making the cell produce ethanol and water. In the presence of oxygen, pyruvate is consumed into CO2 and water by the Krebs cycle at the yeast mytochondria (Figure 2) (van Dijken, J. R., Weusthuis, R. A., Pronk, J. T., 1993, Kinetics of growth and sugar consumption in yeasts, Antonie van Leeuwenhoek 63: 343-352).

When yeasts are grown in the presence of oxygen under low sugar content, or when sugar is slowly uptaken by the cells, the pyruvate dehydrogenase enzyme complex forwards the glycolytic flow to breathing, causing cell replication and, under this situation, Nicotinamide Adenine Dinucleotide (NADH) molecules produced during glycolysis may give their electrons to the electron transport system having oxygen as the final electron acceptor. This not only results in NAD regeneration, but may also cause the synthesis of additional ATP molecules. NADH is formed by redox reaction, is extremely important and acts as an electron transporter during glycolysis. That molecule should be reconstituted to guarantee the continuity of the glycolytic pathway (Salvato, Flavia, 2010, Fermentagao de mosto industrial por linhagens de Saccharomyces cerevisiae com transportador de sacarose e sobre expressao de invertase interna: estudo comparativo com linhagens com alta e baixa atividade de invertase externa, Master’s Degree Thesis, University of Sao Paulo).

In the lack of oxygen or under high sugar concentrations, fermentation is benefited, since the exceeding carbon as not directed to breathing is metabolized by pyruvate decarboxylase (Postma, E., Scheffers, A. W., van Dijken, J. P. (1989), Kinetics of growth and glucose transport in glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066, Yeast, 5 (3), 159-165; van Dijken, J. P., Scheffers, W. A. (1986), Redox balances in the metabolism of sugars by yeasts, FEMS Microbiol. Rev. 32: 199-224).

Gene expression control is a basic regulating pathway for living organisms. In microorganisms, glucose or other carbon sources which may be metabolized suppress the expression of genes coding enzymes related to the metabolism of other sources of carbon. This phenomenon, known as catabolic repression, allows microorganisms to efficiently deal with changes in the carbon sources present for their consumption. In reply to the presence of glucose or sucrose, as well as for the conditions of the medium where the yeast is located, the main repression pathways negatively regulate various genes involved in the absorption and metabolism of carbohydrates, as well as genes involved in gluconeogenesis and breathing. Breathing repression by glucose and sucrose, a phenomenon known as Crabtree effect, may seem counterproductive, but, although breathing is a more efficient method to produce energy, fermentation has the advantage of producing ethanol, thus hindering the growth of competing microorganisms. Furthermore, glucose concentrations and possibly the levels of glycolytic intermediates regulate the expression of various glucose transporters and some glycolytic genes (Figure 3) (Carlson, M., 1999, Glucose repression in yeast, Curr. Opin. Microbiol. 2, 202-207; Gancedo, J. M., 1998, Yeast carbon catabolite repression, Microbiol. Mol. Biol. Rev. 62, 334-361 ; Johnston, M., 1999, Feasting, fasting and fermenting - glucose sensing in yeast and other cells, Trends Genet. 15, 29-33; Verstrepen K. J. et al, 2004, Glucose and sucrose: hazardous fast-food for industrial yeast?, Trends in Biotechnology, vol. 22, n° 10).

Therefore, the main glucose repression route ensures that the preferred sugars are metabolized before the consumption of alternative carbohydrates, such as maltose and galactose. Glucose also reduces fructose absorption, since both sugars are imported by the same transporters, which have higher affinity to glucose than to fructose. Besides that competitive fructose absorption inhibition, recent research shows that glucose may suppress the expression of specific fructose transporters. Besides regulating the uptake of alternative sugars, the main route of glucose repression avoids futile cycles in carbohydrate metabolism, turning off de novo synthesis of glucose by gluconeogenic pathways. In an industrial context, one of the most well known effects of the main glucose repression pathway to the yeast performance is shown by the production of baker’s yeast, during which the change from breathing to fermentation as induced by glucose or sucrose causes sharp reduction in biomass yielding. To avoid these effects, yeasts are mainly grown in well-fed and aerated discontinuous reactors, in which glucose and sucrose concentrations are constantly kept below the concentration limit to induce the Crabtree effect. As mentioned above, besides catabolic repression, glucose and sucrose also entail the activation of a pathway to reduce stress resistance for yeast cells. During industrial processes, yeast cells face various stress conditions, including shear stress; changes in oxygen levels, temperature, osmolarity, pH, ethanol and carbon dioxide concentrations; and possible nutrient imbalance. When the extracellular glucose is exhausted, gene repression is cancelled. Therefore, glucose repression is relieved and alternative carbon sources may be absorbed (Attfield, P. V., 1997, Stress tolerance: the key to effective strains of industrial baker’s yeast, Nat. Biotechnol. 15, 1351-1357; Bauer, F. F., Pretorius, I. S., 2000, Yeast stress response and fermentation efficiency: how to survive the making of wine - a review, S. Afr. J. Enol. Vitic. 21 , 27-51 ; Berthels, N. J. et al, 2004, Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains,

FEMS Yeast Res. 4, 683-689; De Vit, M. J. et al, 1997, Regulated nuclear translocation of the Mig1 glucose repressor, Mol. Biol. Cell 8, 1603-1618; Kim, S. R. et al, 2012, Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol, Trends in Biotechnology, Vol. 30, n° 5; Ostling, J., Ronne, H., 1998, Negative control of the Mig1 repressor by Snf1 -dependent phosphorylation in the absence of glucose, Eur. J. Biochem. 252, 162-168; Sousa, H. R. et al, 2004, Differential regulation by glucose and fructose of a gene encoding a specific fructose/HC symporter in Saccharomyces cerevisiae, Yeast 21 , 519-530; Gongalves, P. et al, 2000, FSY1, a novel gene encoding a specific fructose/HC symporter in the type strain of Saccharomyces carlsbergensis, J. Bacteriol. 182, 5628-5630; Valentinotti, S. et al, 2003, Optimal operation of fed-batch fermentations via adaptive control of overflow metabolite, Control Eng. Pract. 11 , 665-674).

In the yeast S. cerevisiae, the invertase enzyme hydrolyzing sucrose into glucose and fructose is codified by genes of the SUC family, wherein SUC2 is the most common gene. SUC2 regulation occurs in the transcriptional level and the expression is solely controlled by glucose repression mechanisms, but also by an increase in the degradation rate of the respective proteins. Therefore, the use of sucrose is suppressed when glucose or another sugar is present in the growth medium. The repression rate caused by glucose may reach close to 800 times less invertase in yeast in the presence of glucose (Carlson, M., 1987, Regulation of sugar utilization in Saccharomyces species, J. Bacteriol. 169: 4873-4877; Gancedo, J. M., 1992, Carbon catabolite repression in yeast, Eur. J. Biochem. 206, 297-313; Trumbly, R. J., 1992, Glucose repression in the yeast Saccharomyces cerevisiae, Mol. Microbiol. 6: 15-21).

Also known as p-D-fructofuranosidase, invertase enzyme hydrolyzes the glycosidic link for carbohydrates having a non-substituted p-fructofuranosyl radical, being sucrose the preferred substrate (Marquez, L. D. S. et al, 2007, Otimizagao da imobilizagao de invertase por adsorgao em resina de troca ionica para a hidrolise de sacarose, Journal Molecular Catalysis B: Enzymatic, vol. 51 , pages 86-92). The reaction route is based on the formula below, having invertase as the catalyst:

C12H22O11 + H2O + H⁺ invertase C16H12O6 (glucose) + C6H12O6 (fructose) + H⁺

Particularly in S. cerevisiae, there are two forms of invertase: (i) the form located on the wall cell (270 kDa; linked to mannane), mainly occurring as a dimer, tetramer and octamer; and (ii) the intracellular form located within the cytoplasm (135 kDa). Foreign invertase, representing 95% of all yeast invertases, is submitted to catabolic repression by hexoses, while the internal invertase is not (Simionescu, C. et al, 1987, Immobilization of invertase on the Diazonium Salt of 4-Amino Benzoylcellulose, Biotechnol. Bioeng. 29: 361-365; Vitolo, M., Topicos de Enzimologia Industrial, in: Enzimas como agentes biotecnologicos, Said, S. and Pietro, R. C. L. R. Eds, Ed. Legis Summa, Ribeirao Preto, Brazil, 2004; Vitolo, M. 2021. Overview on invertase, World Journal of Pharmacy and Pharmaceutical Sciences, Vol. 10, n° 10, 49-73).

Yeast fermentation occurs, in average, at 30 °C, which is between the temperature to obtain maximum biomass yield in sugar (28.5 °C) and the temperature of maximum growth rate (32 °C). In the process of the invention, the fermentation temperature is thus preferably between 28.5 °C and 33 °C, more preferably between 30 °C and 32 °C. The highest growth rate is reached for pH between 4.0 and 5.5 for the culture medium. The value used for most of the fermentation process is close to pH 4. Near the end of fermentation, after between 12 and 14 hours, pH is increased to about 5.5, for quality requirements. In the process of the invention, pH is thus preferably between pH 4.0 and 5.5, more preferably between pH 4.0 and 5. Anti-lathering agents (preferably natural) may be added to reduce the surface tension as a function of proteins in the fermentation medium (Perez-Torrado, R. et al, 2015, Yeast biomass, an optimized product with myriad applications in the food industry, Trends in Food Science & Technology, 46 (2), 167- 175; Roseiro, C. I. S., Baptista M. S. C. P., 2012, Dimensionamento de uma unidade de produgao de leveduras para a panificagao, Master’s Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar; Vanzella, E. et al, 2014. Processo fermentative na Industrie sucroalcooleira, Acta Iguazu, vol. 3, n° 1 , pages 50-58).

Another aspect of the leaven production process is the supply of air during the fermentation step, which plays an important role for the biomass production process, especially when high yielding is required. Usually, ratios between 0.5 and 2.5 volumes of air per volume of must per minute (wm) are noted and fed batch feeding is used to solve catabolic repression problems. As previously disclosed, the generation of ethanol is inhibited by the presence of oxygen, and thus, cells use oxidative metabolic pathways, with more rational glucose consumption and, consequently, cell growth is higher. On the other hand, even under intense aeration conditions, yeast follows fermentative metabolism to generate ethanol in excessive presence of glucose. Said critical value was determined below 110 mg/l (glucose + fructose) during the leaven production process, under molasses feed (Enfors, S. O., Hedenberg, J., Olsson, K., Simulation of the dynamics in the Baker's yeast process, Bioproc. Eng. 5 (1990), 191 ±198; Crocomo, O. J., Gutierrez, L. E., Caminhos metabolicos, in: Borzani, W., Schmidell, W., Lima, U. A., Aquarone, E., Biotecnologia Industrial, vol. 1 : Fundamentos, Editora Edgard Blucher Ltda., Sao Paulo, 2001 ; Gutierrez, L. E., Acumulo de trealose em linhagens de Saccharomyces durante fermentagao alcoolica, Anais da Escola Superior de Agriculture “Luiz de Queiroz”, vol. 47, n° 2, pages 597-608, 1990; Trevizam, C. J., Molena, C., Dicesar Correia, D., 2013, Estudo dos principals parametros de controle e a agao da trealose no processo de produgao de levedura de panificagao, Revista Engenho, vol. 8). In the process of the invention, the supply of air is thus between 0.1 and 2.5 wm, preferably between 0.2 and 2 wm.

The present invention also refers to a system to perform the process for producing leaven on industrial scale, being thus related to the process steps as disclosed. Therefore, the embodiments and disclosures prepared for the process are also respectively valid for the system.

Thus, the system of the invention comprises at least one aerobic fermentation unit (2) for which a source of carbon comprising hydrolyzed sucrose is provided, as a carbon source for the production of leaven (Figure 5). Preferably, the system also comprises at least one invertase enzyme production unit (41 , 42), e. g. a full mixture reactor, which may (41) or not (42) be connected to the industrial productive chain. Steps (i) to (v) of the process for obtaining invertase as previously disclosed preferably occur in said reactor.

The system of the invention preferably also comprises at least one storage and/or treatment unit for the carbon source (5), comprising sucrose, to which the invertase enzyme is provided, and where the hydrolysis of the sucrose to be used as carbon source for leaven in the aerobic fermentation unit (2) for leaven production takes place. Preferably, at least one storage and/or treatment unit for the carbon source (5) comprises at least one storage tank for molasses (stock) (51), which is usually treated (e. g. clarified) by usual and well-known techniques in the literature (Reed, G., Nagodawithana, W. T., Yeast Technology, Van Nostrand Reinhold, 2^nd ed., 1991 ; Roseiro, C. I. S., Baptista, M. S. C. P., 2012, Dimensionamento de uma unidade de produqao de leveduras para a panificaqao, Master’s Degree in Chemical Technology, Escola Superior de Tecnologia de Tomar; Trevizam, C. J., Molena, C., Dicesar Correia, D., 2013, Estudo dos principals parametros de controle e a aqao da trealose no processo de produqao de levedura de panificaqao, Revista Engenho, vol. 8; White, J., Yeast Technology, John Wiley and Sons Inc., 1^st ed., New York, 1954) and follows to at least another tank (52) for pasteurization of said raw material, so to produce must at least in a third tank (53). Preferably, the enzymatic treatment with invertase is performed in the molasses tank (52) after said treatment and/or in the must tank (53).

The system of the invention preferably also comprises at least one leaven propagation unit (1), where step (b) of the process of the present invention takes place, e. g. a seed tank, which will receive the initial leaven inoculate as previously prepared; at least one separation and/or purification unit (3) (where step (d) of the process of the invention takes place), being preferably a centrifuge; and/or at least one filtering, filtering and pressing, or filtering, pressing and drying unit (6), (where step (e) of the process of the invention takes place). Preferably, in an embodiment of the system of the invention, at least one leaven propagation unit (1) is connected to at least one aerobic fermentation unit (2), which, on the other hand, is connected to both at least one storage and/or treatment unit for the carbon source (5) and at least one separation and/or purification unit (3), which is connected to at least one filtering, filtering and pressing, or filtering, pressing and drying unit (6).

Preferably, the system works as follows: sucrose present in the carbon source for at least one storage and/or treatment unit (5) is hydrolyzed by the invertase enzyme received from at least one invertase enzyme production unit (41 , 42), which is preferably connected to the industrial productive chain (41) (but may also be from an external source, e. g. commercial or specifically produced to provide the chain at issue (42), or any two or all three embodiments may occur in the same process), wherein the carbon source comprising hydrolyzed sucrose from at least one storage and/or treatment unit for the carbon source (5) is then used as a source of carbon for leaven in at least one aerobic fermentation unit (2) to produce leaven, which is separated and/or purified in at least one separation and/or purification unit (3) and filtered, filtered and pressed, or filtered, pressed and dried in at least one filtering, filtering and pressing, or filtering, pressing and drying unit (6); wherein leaven from at least one aerobic fermentation unit (2) was previously propagated in at least one leaven propagation unit (1), after receiving leaven as previously prepared from the initial inoculate. Finally, the present invention refers to the use of a source of carbon comprising hydrolyzed sucrose as carbon source for the aerobic fermentation step for leaven, in the process for producing leaven on industrial scale of the invention, and/or in the system to perform the process for producing leaven on industrial scale of the invention.

Preferred embodiments and characteristics of the process of the invention, as disclosed above, may be equally established for the present system and use of the invention and vice-versa, even if said relationships have not been explicitly indicated.

The invention may be better understood based on the non-limitative examples as disclosed below.

Examples

Example 1

Evaluation of ART conversion rate into leaven by using a source of carbon comprising hydrolyzed sucrose - laboratory scale:

In the present trial, an enzymatic inversion between must and molasses from commercial sugar cane was performed, as the raw material source of carbon, to produce leaven. Hydrolysis of the sucrose as present in the raw material was performed by the invertase enzyme, which was obtained by the conventional production method of enzymatic extract from leaven, as disclosed by the document BR 10 2018 010441 1 , herein incorporated as reference.

Particularly, commercial molasses (80 °Brix) and must produced from said molasses (40 °Brix) were submitted to enzymatic hydrolysis under the temperature of 55 °C and pH 4.5 for 24 hours. The quantity of enzyme as used was 4 kg of extract per ton of sucrose.

Inversion rates for the samples were measured by titration in a Reductec® device, as per manufacturer’s instructions. Reducing sugar (AR) and full reducing sugar (ART) for each sample were measured before inversion (time 0), after 12 h hydrolysis and after 24 h inversion. The ratio from reducing sugar (AR) to full reducing sugar (ART) indicates the inversion rate per time.

The results showed that, within 24 houras, 99.5% sucrose present in both carbohydrate sources, molasses and must were hydrolyzed, producing glucose and fructose (Figure 6).

We should highlight that the inversion percentage for molasses and must may be adjusted for said established rate and inversion time as required, just adjusting the quantity of enzyme as added in the process.

To evaluate the influence of inverted molasses/must in the ART conversion rate of baker’s yeast in comparison with fermentation in crude molasses/must, commercial Fleischmann leavens were used (Saccharomyces cerevisiae . “Sweet Dough” and “Salty Dough” (AB Brasil). For this reason, a culture medium for yeast was prepared (2 g/l yeast extract; 3 g/l (NF ^SC , 3 g/l KH2PO4; 0.3 g/l CaCI₂; 0.3 g/l MgSC ; 1 g/l NaCI and 1 ml trace elements: 1.5 g/l H3BO3; 1 g/l CaCI₂.6H₂O; 0.5 g/l ZnSC>4.7H₂O; 0.15 g/l MnCI₂.4H₂O; 0.15 g/l Na₂MoC>4.2H₂0; 0.1 g/l NiCI₂.6H₂O and 0.05 g/l CUSC>4.5H₂O) and sterilized under autoclave for 30 min at 120 °C. Crude must, inverted must, crude molasses and inverted molasses were heated in water bath at 80 °C for 30 min and used as carbon sources. All manipulations were made in a laminar flow chamber.

The culture medium was added to different carbon sources (the concentration of the carbon source in fermentation was established at 6% total reducing sugars for all treatments) and received an inoculate of each kind of leaven (20 ml pure culture of Fleischman leaven “Sweet Dough” and “Salty Dough”, precultivated for 24 h in the same culture medium). The treatments are as follows:

1. culture medium + must (carbon source) (control 1);

2. culture medium + inverted must (carbon source) (control 2);

3. culture medium + molasses (carbon source) (control 3);

4. culture medium + inverted molasses (carbon source) (control 4);

5. culture medium + crude must + salty dough leaven;

6. culture medium + inverted must + salty dough leaven;

7. culture medium + crude molasses + salty dough leaven;

8. culture medium + inverted molasses + salty dough leaven;

9. culture medium + crude must + sweet dough leaven; and

10. culture medium + inverted must + sweet dough leaven.

The culture was kept by agitation in a rotating shaker at 120 rpm, 28 °C for 48 h. At each 8 h, a portion of the culture was collected for each treatment, centrifuged at 8,000 rpm for 2 min and the wet leaven dough was measured by weighing on analytical scales. The experiments were performed in duplicate.

In the salty dough leaven, results showed that the yeast cultivated in enzymatically inverted must presented 33.3% increase in wet dough in comparison to the same yeast as cultivated in crude must (Figure 7). Inverted must conversion into wet biomass was 1 :1 (1.0 g wet biomass/1.0 g ART in inverted must), while crude must presented conversion of 0.75:1 (0.75 g wet biomass/1 .0 g ART in crude must).

On the other hand, when the carbon source was inverted molasses, there was an even higher increase of 83%, in comparison to the same non-inverted source (Figure 8), considering the maximum points for each case. The conversion from inverted molasses into wet biomass was 0.92:1 (0.92 g wet biomass/1.0 g ART in inverted molasses), while crude molasses had a conversion rate of 0.5:1 (0.5 g wet biomass/1.0 g ART in inverted molasses).

In both experiments, with the use of inverted must or inverted molasses, an increase in the conversion rate of reducing sugars into leaven was observed by time of cultivation, in comparison to the same noninverted sources. Unexpectedly, those increases as observed represent an important and disruptive step forward for the leaven industry. Experiments with “Sweet Dough” leaven were performed in crude and inverted must, indicating that the yeast cultivated in enzymatically inverted must presented 100% more wet dough in comparison to the same yeast cultivated in crude must (Figure 9). The rate of conversion from inverted must into wet biomass was 1 :1 (1.0 g wet biomass/1.0 g ART in the inverted must), while the crude must had a conversion rate of 0.5:1 (0.5 g wet biomass/1 .0 g ART in crude must).

Jointly analyzed, the results showed that the use of inverted carbon sources brought considerable and unexpected increase to the conversion rate in wet leaven dough, in comparison to the use of crude sources, since the same quantity of raw material enabled us to obtain more biomass, a commercial product of the yeast companies.

Although the essays performed in laboratory have been conclusive and consistent on the increase of conversion into leaven with the use of carbon source comprising hydrolyzed sucrose, it was interesting to disclose the implementation of the invention on industrial scale.

Example 2

Evaluation of ART conversion rate into leaven by using a source of carbon comprising hydrolyzed sucrose - pilot industrial scale:

For the present assay, 60 I of cultivation medium were prepared with the following composition: 1.45 kg of crude molasses or 1.45 kg of inverted molasses (99% inversion), 2.3 g/l ammonium sulfate and 0.45 g/l dibasic potassium phosphate. Molasses sucrose hydrolysis was performed at the temperature of 55 °C and pH 4.5 for 24 h. The quantity of enzyme extract as used for molasses hydrolysis was 4 kg conventional enzymatic extract per ton of sucrose. The leaven used to obtain the enzyme (Saccharomyces cerevisiae commercial “Sweet Dough” from Fleischmann) was from the industrial production process for commercial leaven, as indicated by Figure 5. Therefore, a portion (4 kg commercial leaven in 500 g tablets/ton of sucrose) of the produced biomass, after the biomass separation process by conventional centrifugation, followed to a tank, where it was treated until reaching the enzymatic complex as disclosed by the previous example. The product comprising invertase followed to the molasses tank for sucrose hydrolysis, from which the must was produced and said raw material was used in the fermentation tank. Fermentation was performed in a 100 I hermetic jacketed stainless steel reactor, with a shaker and air sintered air diffuser made of stainless steel. The growth was performed with air injection at 0.5 wm (air volume/cultivation medium volume/minute) within the first four hours of cultivation and 1.0 wm in the following hours, using a 0-150 l/min rotameter. The cultivation medium was sterilized in the reactor at 85 °C for 40 minutes. The test was performed with leaven at 32 °C for 12 hours. A sample was collected at each hour of fermentation, and the biomass was measured by weight on analytic scales.

The performance of yeast biomass in crude and inverted molasses is shown by Figure 10. By using inverted molasses, the maximum biomass reached 27.5 g/l at the highest growth point (11 hours of cultivation), while crude molasses reached maximum biomass of 20 g/l at the maximum growth point (between 8 and 9 hours of cultivation). Considering that the molasses have in average 55% available sucrose, i. e. 0.78 kg fermentable sugar (ART), the rate of conversion into biomass was 1 :2.07 for inverted molasses, i. e. 0.798 kg of sugar generated 1.65 kg of wet biomass (27.5 g/l x 60 g/l) and 1 :1.5 for crude molasses, i. e. 0.798 kg sugar (ART) generated 1.2 kg wet biomass (20 g/l x 60 I). Therefore, the use of inverted molasses increased biomass production in 37% over crude molasses at the maximum point of each biomass.

This assay was repeated, under the same conditions, except for the quantity of crude molasses or inverted molasses (99% inversion), which was 6 kg, and the growth of biomass performed with fixed air injection of 12 l/min (0.2 wm). The performance results for yeast biomass in crude and inverted molasses are shown on Figure 11. By using inverted molasses, the maximum biomass reached 55 g/l within 8 hours of cultivation, while crude molasses reached maximum biomass of 35 g/l within 6 hours of cultivation. Considering that the molasses have in average 55% available sucrose, i. e. 3.3 kg fermentable sugar in the present case, the rate of conversion into biomass was 1 :1 for inverted molasses, i. e. 3.3 kg sugar generated 3.3 kg wet biomass (55 g/l x 60 g/l) and 1 :0.64 for crude molasses, i. e. 3.3 kg sugar (ART) generated 2.1 kg wet biomass (35 g/l x 60 I). Therefore, the use of inverted molasses increased biomass production in 57% over crude molasses at the maximum point of each biomass.

The industrial reference on ART conversion into wet biomass is 0.665 kg ART/1.0 kg wet biomass (https://www.vogelbusch-biocommodities.com/technology/yeast-process-plants/bakers-yeast-technology/, accessed on September 20, 2022), i. e., according to the obtained results, the crude molasses in the present assay on industrial scale reproduced the reference literature in the industry. However, with the use of inverted molasses, the conversion was much higher.

We should highlight that this assay was also performed, within the conditions as mentioned, with a commercial line of Saccharomyces cerevisiae “Sweet Dough” from the company Itaiquara, with equivalent results, indicating that the advantageous results disclosed by the present invention do not depend on the strain as used (i. e. they can be applied to any kind of leaven/yeast), or on the carbon source as used.

We suggest that the presence of glucose monosaccharide promotes catabolic repression, avoiding the yeast from wasting energy, producing molecules involved in sucrose hydrolysis and others, thus saving cell energy and taking the cell to direct the energy flow to biomass production, and thus increase the conversion of substrate into biomass.

Jointly, the results indicate that there is higher conversion of reducing sugars into leaven, also for the industrial production of leaven, when the aerobic fermentation of yeast is performed with a carbon source comprising hydrolyzed sucrose as a source of carbon.

Claims

1 . Process for producing leaven on industrial scale, characterized by comprising the supply, as a carbon source in the aerobic fermentation step to produce leaven, of a source of carbon comprising hydrolyzed sucrose.

2. Process, of claim 1 , characterized by sucrose hydrolysis being performed by enzymatic inversion, chemical inversion and/or resins.

3. Process, of any of claims 1 or 2, characterized by the enzymatic inversion being performed by invertase, which origin may be external to the industrial productive chain, such as commercial origin, and/or may be specifically produced for supply to the productive chain at issue, and/or obtained from the leaven produced by its own industrial productive chain.

4. Process, of any of claims 1 to 2, characterized by the chemical inversion being performed with one or more acids, wherein one or more acids are selected from the group consisting of: hydrochloric acid, phosphoric acid, sulfuric acid, citric acid and lactic acid.

5. Process, of any of claims 1 to 4, characterized by comprising the steps of: a. preparing the initial leaven inoculate; b. propagating the leaven prepared in (a); c. promoting the aerobic fermentation of the leaven prepared in (b) for leaven production, wherein the carbon source comprising hydrolyzed sucrose is provided as a source of carbon; d. separating and/or purifying the product as obtained in (c) to concentrate the leaven; and e. filtering, filtering and pressing, or filtering, pressing and drying the product obtained in (d).

6. Process, of any of claims 1 to 5, characterized by the leaven being yeast, more preferably from genus Saccharomyces spp., even more preferably from the species Saccharomyces cerevisiae.

7. Process, of any of claims 1 to 6, characterized by sucrose being present in the source of carbon as used for fermentation, wherein said source is selected from the group consisting of granulated sugar, pure sucrose, VHP (Very High Polarization) sugar, VVHP (Very Very High Polarization) sugar, demerara sugar, brown sugar, concentrated sugar cane juice, molasses and must, or any of their combinations, even if said sources are preferably derived from sugar cane or sugar beets.

8. Process, of any of claims 1 to 7, characterized by between 50 and 100% sucrose being hydrolyzed, preferably between 85 and 99.9%, more preferably between 90 and 99.9%.

9. Process, of any of claims 1 to 8, characterized by aerobic fermentation being performed with air supply between 0.1 and 2.5 wm, temperature between 28.5 °C and 34 °C and pH between 4.0 and 5.5.

10. Process, of any of claims 1 to 9, characterized by sucrose as carbon source having more than 1 °Brix, preferably between 1 and 99 °Brix, more preferably between 15 and 80 °Brix.

11. Process, of any of claims 1 to 10, characterized by the leaven production process being a process causing an increase in ART conversion into leaven at the end of the aerobic fermentation step.

12. System to perform the process for producing leaven on industrial scale, of any of claims 1 to 11 , characterized by comprising at least one aerobic fermentation unit (2) to which, as a source of carbon for the leaven, a carbon source comprising hydrolyzed sucrose is provided.

13. System, of claim 12, characterized by also comprising at least one invertase enzyme production unit (41 , 42) which may (41) or not (42) be connected to the industrial productive chain.

14. System, of any of claims 12 to 13, characterized by also comprising at least one storage and/or treatment unit for a carbon source (5) comprising sucrose, to which invertase enzyme is provided, where the hydrolysis of sucrose to be used as a carbon source for leaven in the aerobic fermentation unit (2) to produce leaven will take place.

15. System, of any of claims 12 to 14, characterized by also comprising: at least one propagation unit for leaven (1), which will receive leaven as previously prepared from the initial inoculate; at least one separation and/or purification unit (3); and/or at least one unit for filtration, filtration and pressing, or filtration, pressing and drying (6).

16. System, of any of claims 12 to 15, characterized by at least one leaven propagation unit (1) being connected to at least one aerobic fermentation unit (2) which, on the other hand, is connected to both at least one storage and/or treatment unit for a carbon source (5) and to at least one separation and/or purification unit (3), which is connected to at least one unit for filtering, filtering or pressing, or filtering, pressing and drying (6).

17. System, of any of claims 12 to 16, characterized by: the sucrose present in the carbon source of at least one storage and/or treatment unit (5) being hydrolyzed by the invertase enzyme received from at least one production unit for invertase enzyme (41 , 42), which is preferably connected to the industrial productive chain (41), wherein the source of carbon comprising hydrolyzed sucrose in at least one storage and/or treatment unit (5) is then used as a source of carbon for leaven in at least one aerobic fermentation unit (2) for producing leaven, so to produce leaven biomass, which is separated and/or purified in at least one separation and/or purification unit (3) and filtered, filtered and pressed, or filtered, pressed and dried in at least one unit for filtering, filtering and pressing, or filtering, pressing and drying (6); wherein the leaven of at least one aerobic fermentation unit (2) was previously propagated in at least one leaven propagation unit (1), after receiving the leaven as previously prepared from the initial inoculate.

18. Use of a carbon source comprising hydrolyzed sucrose, characterized by being as a source of carbon in the aerobic fermentation step in a process for producing leaven on industrial scale of any of claims 1 to 11 , and/or in a system to perform the process for producing leaven on industrial scale of any of claims 12 to 17.