Microbial protein production from lactose-rich effluents through food-grade mixed cultures: Effect of carbon to nitrogen ratio and dilution rate.

Overabundant agro-industrial side streams such as lactose-rich effluents from dairy activities offer multiple valorisation opportunities. In the present study, a food-grade mixed culture of bacteria and yeasts was tested under different operational conditions for the treatment and the valorisation of cheese whey permeate (CWP), the residue of whey protein recovery, into microbial protein (MP). Under continuous aerobic fermentation settings, the carbon-to-nitrogen (C/N) ratio showed little to no influence on the system performances and MP quality as compared to dilution rates (D), leading to a final protein content as high as 76%. Under high D values, instead, while biomass productivity increased, N-efficiency and protein content decreased. Unlike the bacterial community, the yeast one proved to be highly stable and less influenced by the increase of D. A preliminary estimate indicated that 2-11% of the future MP-based food production could be satisfied by only valorising lactose-rich dairy residues such as CWP.

Recovery of lactose from simulated delactosed whey permeate by a low-temperature crystallization process.

Delactosed whey permeate is the mother liquor/by-product of lactose manufacture, but it still contains around 20 wt% lactose. The high mineral content, stickiness, and hygroscopic behavior prevent further recovery of lactose in the manufacturing process. Therefore, its use is currently limited to low-value applications such as cattle feed, and more often it is seen as waste. This study investigates a new separation technique operating at sub-zero conditions. At low temperature, precipitation of calcium phosphate is expected to be reduced and the lower solubility at sub-zero temperature makes it possible to recover a large portion of the lactose. We found that lactose could be crystallized at sub-zero conditions. The crystals had a tomahawk morphology and an average size of 23 and 31 µm. In the first 24 h, the amount of calcium phosphate precipitated was limited, whereas the lactose concentration was already close to saturation. The overall rate of crystallization was increased compared with the crystals recovered from a pure lactose solution. Mutarotation was rate limiting in the pure system but it did not limit the crystallization of lactose from delactosed whey permeate. This resulted in faster crystallization; after 24 h the yield was 85%.

Effect of Milk Protein and Whey Permeate in Large-Quantity Lipid-Based Nutrient Supplement on Early Child Development among Children with Stunting: A Randomized 2 × 2 Factorial Trial in Uganda.

Stunting affects 22% children globally, putting them at risk of adverse outcomes including delayed development. We investigated the effect of milk protein (MP) vs. soy and whey permeate (WP) vs. maltodextrin in large-quantity, lipid-based nutrient supplement (LNS), and LNS itself vs. no supplementation, on child development and head circumference among stunted children aged 1-5 years. We conducted a randomized, double-blind, community-based 2 × 2 factorial trial in Uganda (ISRCTN1309319). We randomized 600 children to one of four LNS formulations (~535 kcal/d), with or without MP (n = 299 vs. n = 301) or WP (n = 301 vs. n = 299), for 12 weeks or to no supplementation (n = 150). Child development was assessed using the Malawi Development Assessment Tool. Data were analyzed using linear mixed-effects models. Children had a median [interquartile range] age of 30 [23; 41] months and mean ± standard deviation height-for-age z-score of -3.02 ± 0.74. There were no interactions between MP and WP for any of the outcomes. There was no effect of either MP or WP on any developmental domain. Although LNS itself had no impact on development, it resulted in 0.07 (95%CI: 0.004; 0.14) cm higher head circumference. Neither dairy in LNS, nor LNS in itself, had an effect on development among already stunted children.

Design of a stable ethanologenic bacterial strain without heterologous plasmids and antibiotic resistance genes for efficient ethanol production from concentrated dairy waste.

Engineering sustainable bioprocesses that convert abundant waste into fuels is pivotal for efficient production of renewable energy. We previously engineered an Escherichia coli strain for optimized bioethanol production from lactose-rich wastewater like concentrated whey permeate (CWP), a dairy effluent obtained from whey valorization processes. Although attractive fermentation performances were reached, significant improvements are required to eliminate recombinant plasmids, antibiotic resistances and inducible promoters, and increase ethanol tolerance. Here, we report a new strain with chromosomally integrated ethanologenic pathway under the control of a constitutive promoter, without recombinant plasmids and resistance genes. The strain showed extreme stability in 1-month subculturing, with CWP fermentation performances similar to the ethanologenic plasmid-bearing strain. We then investigated conditions enabling efficient ethanol production and sugar consumption by changing inoculum size and CWP concentration, revealing toxicity- and nutritional-related bottlenecks. The joint increase of ethanol tolerance, via adaptive evolution, and supplementation of small ammonium sulphate amounts (0.05% w/v) enabled a fermentation boost with 6.6% v/v ethanol titer, 1.2 g/L/h rate, 82.5% yield, and cell viability increased by three orders of magnitude. Our strain has attractive features for industrial settings and represents a relevant improvement in the existing ethanol production biotechnologies.

Valorisation of Whey Permeate in Sequential Bioprocesses towards Value-Added Products-Optimisation of Biphasic and Classical Batch Cultures of Kluyveromyces marxianus.

Whey permeate is categorised as hazardous wastewater for aquatic environments, mainly due to its high lactose content. Therefore, it must be valorised before being released into the environment. One pathway for whey permeate management is its use in biotechnological processes. Herein, we present roads for whey permeate valorisation with the K. marxianus WUT240 strain. The established technology is based on two bioprocesses. During first, 2.5 g/L 2-phenylethanol and fermented plant oils enriched with different flavourings are obtained after 48 h biphasic cultures at 30 °C. The second process leads to a maximum of 75 g ethanol/L (YP/S = 0.53 g/g) after 96 h at 30 °C. Moreover, established whey permeate valorisation pathways reduced its biochemical oxygen demand and chemical oxygen demand values by 12- to 3-fold, respectively. Together, the present study reports a complete, effective, and environmentally friendly whey permeate management strategy while simultaneously enabling the acquisition of valuable compounds with substantial application potential.

Scaling up continuous eutectic freeze crystallization of lactose from whey permeate: A pilot plant study at sub-zero temperatures.

Eutectic freeze crystallization is explored as an alternative to the state-of-the-art evaporation process for the recovery of lactose from whey permeate. At the so-called eutectic freezing point, both water (the solvent) and lactose (the solute) crystallize and can be removed continuously while continuously feeding whey permeate. This continuous process is demonstrated on a pilot scale at sub-zero temperatures. In the first instance, only freeze concentration of whey permeate took place at -4 °C. It was possible to reach a lactose concentration of 30 wt% and hardly any nucleation was observed. The resulting ice had high purity, with a lactose concentration of ±2 wt%. Next, the eutectic phase was reached, and lactose and ice crystallized simultaneously and were continuously removed from the system, the resulting crystals had parallelogram morphology with an average size of 10 µm. Ice was recovered at a rate of 60 kg/h and lactose was recovered at a rate of 16 kg/h, yielding over 80% of the feed lactose. A conceptional design was proposed for an improved yield and reduction of energy. Yields of at least 80% and up till 95% could be achieved. Compared to the state-of-the-art mechanical vapor recompression (MVR), EFC is 80% more energy efficient.

Molecular profiling of whey permeate reveals new insights into molecular affinities related to industrial unit operations during lactose production.

Lactose powder production from whey permeate generates various side-streams. Molecular profiling of these side-streams and lactose powder can help to detect minor compounds affecting lactose crystallization, lactose powder properties and document the composition of the underutilized side-streams. In this study, whey permeate, lactose powder and intermediate streams from trial lactose productions were analyzed using gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance (1H NMR) spectroscopy. In total, 110 compounds were identified and 49 were quantified. Linking the molecular profiles to in-process steps revealed differential compositional attenuation by the unit operations. Small molecules (e.g. methanol) and a few larger molecules (e.g. fatty acids) permeated reverse osmosis membrane, while twenty-three compounds (e.g. hydroxypyruvic acid, malonic acid, gluconic acid and ribonic acid) co-crystallized with lactose and ended up in lactose power. These results help to better understand and control lactose powder production and highlights possibilities to develop new food ingredients.

Whey permeate as a phosphorus source for algal cultivation.

Microalgal cultivation for biodiesel and feed requires recycled nutrient resources for a sustainable long-term operation. Whey permeate (WP) from dairy processing contains high organic load (lactose, oils, and proteins) and nitrogen (resources tested for microalgal cultivation) and organic phosphorus (P) that has not yet been tested as a P source for microalgal cultivation. We explored the potential of green algae strains (brackish) and polyculture (freshwater) in exploiting P from WP added to a medium based on either seawater (7 psu) or landfill leachate. Both strains showed a capacity of using organic P in WP with equal growth rates (0.94-1.12 d-1 ) compared with chemical phosphate treatments (0.88-1.07 d-1 ). The polyculture had comparable growth rate (0.25-0.57 d-1 ) and biomass yield (152.1-357.5 mg L-1 ) and similar or higher nutrient removal rate in the leachate-WP medium (1.3-6.4 mg L-1  day-1 nitrogen, 0.2-1.1 mg L-1  day-1 P) compared with the leachate-chemical phosphate medium (1.2-4.7 mg L-1  day-1 nitrogen, 0.3-1.4 mg L-1  day-1 P). This study showed that WP is a suitable P source for microalgal cultivation over a range of salinities. To date, this is the first study demonstrating that raw WP can replace mineral P fertilizer for algal cultivation. PRACTITIONERS POINTS: Whey permeate is a comparable phosphorus source to standard fertilizers used in algal cultivation. Green algae removed phosphorus effectively from whey permeate. Microalgal cultivation is a good approach for treatment of whey permeate in combination with a nitrogen-rich wastewater.

Screening and Scale-up of Nanofiltration Membranes for Concentration of Lactose and Real Whey Permeate.

In dairy industry huge quantities of whey accumulate as a by-product. In particular the containing lactose was not produced profitably in the past. Thus, the trend goes towards modification and sustainable use of lactose for which a concentration step is required. Nanofiltration (NF) has shown to be a good choice since partial demineralization can be realized in parallel. Therefore, in this study, 10 commercial polymer NF membranes were studied in detail and systematically for their suitability to concentrate lactose, with the proviso of high flux and high to complete rejection. Preliminary trials were conducted with flat-sheet membranes and a lactose model solution and the influence of transmembrane pressure (TMP), temperature and lactose concentration was studied. Finally, results were evaluated by using spiral wound modules and real industrial whey permeate. The results offered that a membrane screening is essentially since no correlation between molecular weight cut-off (MWCO) and permeate flow could be found. The conclusions found for the lactose model solution were in good agreement with the whey permeate, but as the ions contribute to the osmotic pressure of the feed the deviations increase in the course of concentration since ions are also partly retained.

Utilization of delactosed whey permeate for the synthesis of ethyl acetate with Kluyveromyces marxianus.

Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

Screening of non-protein nitrogen compounds in lactose refining streams from industrial whey permeate processing.

The NPN compounds from dairy processing side-streams are a promising source for new products. In this study, the NPN profile of lactose production samples was screened using GC-MS and 1H NMR spectroscopy. These analytical platforms allowed the identification of 35 NPN compounds including, amino acids and derivatives, amino alcohols, organic acids, and other classes. Quantification of the NPN compounds revealed their attenuation by unit operations during a trial lactose production. Urea, ammonia, glycerophosphocholine, creatine, creatinine, orotic acid and choline were the most dominant compounds. Mother liquor concentrate had the highest concentration of NPN, whereas lactose powder had substantial relative amounts of N-acetylglucosamine, phosphocholine and orotic acid. The NPN compounds added up to 57-99% of the total nitrogen, depending on the sample type. The highest nitrogen recovery was found for the reverse osmosis retentate, mother liquid concentrate, wash water and reverse osmosis permeate, whereas the lowest was found for lactose powder.

Isolation, identification, and application of lactic acid-producing bacteria using salted cheese whey substrate and immobilized cells technology.

BACKGROUND: Lactic acid bacteria (LAB) could be used for bio-production of lactic acid (LA) from wastes of dairy industries. This study aimed to produce LA using isolated and identified LAB capable of withstanding high salt concentration of salted cheese whey and adopting immobilization technique in repeated batch fermentation process. RESULTS: Seventy four isolates of LAB were isolated from salted cheese whey and examined for lactic acid production. The superior isolates were biochemically and molecularly identified as Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. Then the best of them, Enterococcus faecalis, Enterococcus hirae and dual of them besides Lacticaseibacillus casei were immobilized by sodium alginate 2% in entrapped cells. Repeated batch fermentation was executed for LA production from the mixture of salted whey and whey permeate (1:1) using immobilized strains during static state fermentation under optimum conditions (4% inoculum size in mixture contained 5% sucrose and 0.5% calcium carbonate and incubation at 37 °C). The potent bacterial strain was Enterococcus faecalis which gave the maximum LA production of 36.95 g/l with a yield of 81% after 36 h incubation at 37 °C in presence of 5% sugar. CONCLUSION: Immobilized cells exhibited good mechanical strength during repetitive fermentations and could be used in repetitive batch cultures for more than 126 days.

Spray-Dried Microcapsules of Cheese Whey and Whey Permeate as a Strategy to Protect Chia Oil from Oxidative Degradation.

Research background: Cheese whey and whey permeate are dairy industry by-products usually sent to effluent treatment or incorrectly disposed in the environment, generating costs for the production of dairy products and environmental problems due to the high organic load. Cheese whey and whey permeate can be reused as wall materials to form chia oil microcapsules, which act as a barrier to prooxidants. This study aims to develop an encapsulation method by spray-drying to protect chia oil using dairy by-products as wall materials. Experimental approach: We evaluated cheese whey, whey permeate and mixtures of m(cheese whey):m(whey permeate)=50, 70 and 80% as encapsulating agents with the spray-drying process. Initially, we characterized the chia oil and encapsulating materials. Chia oil emulsions were prepared using the encapsulating materials and an emulsifier. The stability of the emulsions was evaluated by creaming index, and they were characterized according to size distribution and polydispersity index. Emulsions were encapsulated in a spray dryer with inlet and outlet air temperature at 125 and 105 °C, respectively. After encapsulation, we assessed the oxidative degradation of chia oil over 30 days of storage by determining the peroxide index. Results and conclusions: Emulsions presented creaming index between 51 and 83% in all formulations, and the oxidative stability of microencapsulated chia oil was significantly higher than that of free chia oil after 30 days. Wall material combination affected both encapsulation efficiency and oxidation protection. The cheese whey and whey permeate (8:2) mixture exhibited the highest encapsulation efficiency (70.07%) and ability to protect the chia seed oil. After 30 days, the peroxide value was below the maximum limit considered safe for human consumption. Novelty and scientific contribution: According to these results, dairy by-products can be used for encapsulation of oxidation-sensitive oils. This represents an alternative use for dairy by-products, which otherwise are discarded and can impact the environment due to their high organic load. Our findings suggest that dairy by-products can be effectively used as wall materials to generate value-added products.

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L-1 h-1.

Impacts of weaning age on dietary needs of whey permeate for pigs at 7 to 11 kg body weight.

BACKGROUND: Whey permeate is an effective lactose source for nursery pigs and the most benefits are obtained when pigs are at 7 to 11 kg BW. Altering weaning ages could cause different length of early-weaner phases until 7 kg BW and thus it would influence the dietary need of whey permeate during 7 to 11 kg BW of pigs. This study aimed to evaluate if weaning ages would affect the dietary needs of whey permeate for optimum growth performance of pigs at 7 to 11 kg BW. METHODS: A total of 1,632 pigs were weaned at d 21 (d 21.2 ± 1.3) or d 25 (d 24.6 ± 1.1) after birth. All pigs had a common early-weaner feeds until they reached 7 kg BW. When pigs reached 7 kg BW within a weaning age group, they were allotted in a randomized complete block design (2 × 4 factorial). Two factors were weaning age groups (21 and 25 d of age) and varying whey permeate levels (7.50%, 11.25%, 15.00%, and 18.75%). Data were analyzed using the GLM and NLIN procedures of SAS for slope-ratio and broken-line analyses to determine the growth response to whey permeate and optimal daily whey permeate intake for the growth of the pigs weaned at different ages. RESULTS: Pigs weaned at 21 d of age had a common diet for 11 d to reach 7 kg BW whereas pigs weaned at 25 d of age needed 2 d. The G:F of pigs weaned at 25 d of age responded to increased daily whey permeate intake greater (P < 0.05) than pigs weaned at 21 d of age. Breakpoints were obtained (P < 0.05) at 88 and 60 g/d daily whey permeate intake or 17.0% and 14.4% of whey permeate for G:F of pigs weaned at 21 and 25 d of age, respectively. CONCLUSION: Pigs weaned at an older age with a short early-weaner phase had a greater growth response to whey permeate intake compared with pigs weaned at a younger age with a long early-weaner phase. Altering weaning ages affected dietary needs of whey permeate for optimum growth performance of pigs from 7 to 11 kg BW.

Whey permeate as a substrate for the production of freeze-dried Lactiplantibacillus plantarum to be used as a malolactic starter culture.

The aim of this work was to obtain freeze-dried biomass of the native Patagonian Lactiplantibacillus plantarum strain UNQLp 11 from a whey permeate (WP)-based medium and compare it with the growth in commercial MRS broth medium. Survival and activity of the freeze-dried Lb. plantarum strain were investigated after inoculation in wine as a starter culture for malolactic fermentation (MLF). The effect of storage and rehydration condition of the dried bacteria and the nutrient supplementation of wine were also studied. The freeze-dried cultures from WP and those grown in MRS showed similar survival results. Rehydration in MRS broth for 24 h and the addition of a rehydration medium to wine as nutrient supplementation improved the survival under wine harsh conditions and guaranteed the success of MLF. Storage at 4 °C under vacuum was the best option, maintaining high cell viability for at least 56 days, with malic acid consumption higher than 90% after 7 days of inoculation in a wine-like medium. These results represent a significant advance for sustainable production of dried malolactic starter cultures in an environmentally friendly process, which is low cost and easy to apply in winemaking under harsh physicochemical conditions.

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae.

OBJECTIVES: Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus. RESULTS: Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower. CONCLUSIONS: Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

The Role of Milk Protein and Whey Permeate in Lipid-based Nutrient Supplements on the Growth and Development of Stunted Children in Uganda: A Randomized Trial Protocol (MAGNUS).

Stunting is associated with cognitive impairment and later chronic disease. Previous trials to prevent stunting have had little effect, and no trials seem to have provided larger amounts of energy and high-quality proteins to already stunted children. We aimed to assess the effects of milk protein (MP) and whey permeate (WP) in large-quantity lipid-based nutrient supplements (LNS-LQ), among stunted children, on linear growth and child development. This was a randomized, double-blind, 2-by-2 factorial trial. Stunted children aged 12-59 mo from eastern Uganda (n = 750) were randomly assigned to receive 100 g LNS-LQ with or without MP and WP (n = 4 × 150) or no supplement (n = 150) for 3 mo. The primary outcomes were change in knee-heel and total length. Secondary outcomes included child development, body composition, anthropometry, and hemoglobin. Micronutrient status, intestinal function, and microbiota were also assessed. Our findings will contribute to an understanding of the role of milk ingredients and LNS in linear catch-up growth. This trial was registered at www.isrctn.com as ISRCTN13093195.

Whey Protein Derived Mouthdrying Found to Relate Directly to Retention Post Consumption but Not to Induced Differences in Salivary Flow Rate.

Whey protein is fortified into beverages to provide functional benefits, however, these beverages are considered mouthdrying. To date whey protein derived mouthdrying has not been quantified using a 'physical measure' in parallel with rated perception. Saliva flow could also relate to whey protein derived mouthdrying, however this has not been previously tested as an intervention. Accordingly, volunteers (n = 40) tested mouthdrying in different whey beverages and the sensory profile was evaluated by a trained sensory panel (n = 10). Volunteers also rated mouthdrying combined with collection of saliva samples post beverage consumption to measure retention to the oral cavity. To modulate saliva flow rate, volunteers both chewed on parafilm (to increase saliva flow) and used cotton wool (to remove saliva) before tasting beverages and rating mouthdrying. Both the volunteers and sensory panel rated whey protein beverages (WPB) as significantly more mouthdrying than the control beverage (whey permeate). The significantly higher rating of mouthdrying from the volunteers coincided with significantly higher protein concentration in saliva samples post WPB consumption, supporting mucoadhesion as the mechanism. Modulating saliva flow did not lead to any difference in rated mouthdrying and future work would be beneficial to evaluate further the influence of natural variation in salivary flow rate.

Dose-response and functional role of whey permeate as a source of lactose and milk oligosaccharides on intestinal health and growth of nursery pigs.

Two experiments were conducted to evaluate dose-response and supplemental effects of whey permeate on growth performance and intestinal health of nursery pigs. In experiment (exp.) 1, 1,080 pigs weaned at 6.24 kg body weight (BW) were allotted to five treatments (eight pens/treatment) with increasing levels of whey permeate in three phases (from 10% to 30%, 3% to 23%, and 0% to 9% for phase 1, 2, and 3, respectively) fed until 11 kg BW and then fed a common phase 4 diet (0% whey permeate) until 25 kg BW in a 48-d feeding trial. Feed intake and BW were measured at the end of each phase. In exp. 2, 1,200 nursery pigs at 7.50 kg BW were allotted to six treatments (10 pens/treatment) with increasing levels of whey permeate from 0% to 18.75% fed until 11 kg BW. Feed intake and BW were measured during 11 d. Six pigs per treatment (1 per pens) were euthanized to collect the jejunum to evaluate tumor necrosis factor-alpha, interleukin-8 (IL-8), transforming growth factor-beta 1, mucin 2, histomorphology, digestive enzyme activity, crypt cell proliferation rate, and jejunal mucosa-associated microbiota. Data were analyzed using contrasts in the MIXED procedure and a broken-line analysis using the NLIN procedure of SAS. In exp. 1, increasing whey permeate had a quadratic effect (P < 0.05) on feed efficiency (G:F; maximum: 1.35 at 18.3%) in phase 1. Increasing whey permeate linearly increased (P < 0.05) average daily gain (ADG; 292 to 327 g/d) and G:F (0.96 to 1.04) of pigs in phase 2. In exp. 2, increasing whey permeate linearly increased (P < 0.05) ADG (349 to 414 g/d) and G:F (0.78 to 0.85) and linearly increased (P < 0.05) crypt cell proliferation rate (27.8% to 37.0%). The breakpoint from a broken-line analysis was obtained at 13.6% whey permeate for maximal G:F. Increasing whey permeate tended to change IL-8 (quadratic, P = 0.052; maximum: 223 pg/mg at 10.9%), to decrease Firmicutes:Bacteroidetes (P = 0.073, 1.59 to 1.13), to increase (P = 0.089) Bifidobacteriaceae (0.73% to 1.11%), and to decrease Enterobacteriaceae (P = 0.091, 1.04% to 0.52%) and Streptococcaceae (P = 0.094, 1.50% to 0.71%) in the jejunal mucosa. In conclusion, dietary inclusion of whey permeate increased the growth of nursery pigs from 7 to 11 kg BW. Pigs grew most efficiently with 13.6% whey permeate. Improvement in growth performance is partly attributed to stimulating intestinal immune response and enterocyte proliferation with positive changes in jejunal mucosa-associated microbiota in nursery pigs.